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E S T R AT T I R E L A Z I O N I
COMUNICAZIONI BREVI • POSTERS
E S T R AT T I R E L A Z I O N I A Z I E N D A L I
CONGRESS PROCEEDINGS
S H O R T C O M M U N I C AT I O N S • P O S T E R S
C O M PA N Y R E S E A R C H A B S T R A C T S
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SOCIETÀ CULTURALE ITALIANA VETERINARI PER ANIMALI DA COMPAGNIA
SOCIETÀ FEDERATA ANMVI
organizzato da
organized by
Soc. Cons. a r.l.
Azienda con sistema qualità certificato ISO 9001:2008
Company with a certified quality system ISO 9001:2008
CONGRESSO INTERNAZIONALE SCIVAC
LEISHMANIOSI CANINA E ALTRE MALATTIE
TRASMESSE DA VETTORI.
A CHE PUNTO SIAMO?
8-10 MARZO 2013, PISA
E S T R AT T I R E L A Z I O N I
COMUNICAZIONI BREVI • POSTERS
E S T R AT T I R E L A Z I O N I A Z I E N D A L I
INTERNATIONAL SCIVAC CONGRESS
CANINE LEISHMANIOSIS AND OTHER
VECTOR-BORNE DISEASES:
OUR CURRENT STATE OF KNOWLEDGE
March 8th -10th 2013 - PISA - Italy
S H O R T C O M M U N I C AT I O N S • P O S T E R S
C O M PA N Y R E S E A R C H A B S T R A C T S
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COMMISSIONE SCIENTIFICA
CONGRESSUALE
SCIENTIFIC COMMITTEE
Gad Baneth, DVM, PhD,
Dipl ECVCP, Jerusalem, Israel
Walter Bertazzolo, Med Vet,
Dipl ECVCP, Pavia, Italy
Laia Solano Gallego, DVM, PhD,
Dipl ECVCP, Barcelona, Spain
Laura Ordeix, DVM, Dipl ECVD,
Barcelona, Spain
Fulvio Stanga, Med Vet, Cremona, Italy
COORDINATORE CONGRESSUALE
SCIVAC CONGRESS COORDINATOR
MONICA VILLA
Tel: +39 0372 403504
E-mail: commscientifica@scivac.it
SEGRETERIA MARKETING
MARKETING SECRETARY
FRANCESCA MANFREDI
Tel: +39 0372 403538
E-mail: marketing@evsrl.it
SEGRETERIA ISCRIZIONI
REGISTRATION SECRETARY
PAOLA GAMBAROTTI
Tel: +39 0372 403508
Fax: +39 0372 403512
E-mail: info@scivac.it
CONSIGLIO DIRETTIVO SCIVAC
SCIVAC BOARD
Federica Rossi President
Dea Bonello President Senior
Walter Bertazzolo Secretary
Guido Pisani Treasurer
Alberto Crotti Fellow member
David Chiavegato Fellow member
Bruno Peirone Fellow member
ORGANIZZAZIONE CONGRESSUALE
CONGRESS ORGANIZER
EV - Eventi Veterinari - Via Trecchi 20
26100 CREMONA (Italy)
DIRETTORE SCIENTIFICO
SCIENTIFIC CONGRESS DIRECTOR
FULVIO STANGA, Med Vet, Cremona
Soc. Cons. a r.l.
Company with a certified quality system ISO 9001:2008
Società Federata ANMVI
SCIVAC ringrazia gli Sponsor per il sostegno dato all’evento
SCIVAC wishes to thank all the key Sponsors for their support
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CANINE LEISHMANIOSIS AND OTHER VECTOR-BORNE DISEASES: OUR CURRENT STATE OF KNOWLEDGE
MARCH 8TH -10TH 2013 - PISA - ITALY
INVITED SPEAKERS
GAD BANETH
DVM, PhD,
Dipl ECVCP,
Jerusalem, Israel
Prof. Baneth graduated
from the Hebrew University Koret School of
Veterinary Medicine in Israel in 1990.
He did a Small Animal internship
and residency at the Hebrew University until 1994 followed by a fellowship
in Internal Medicine and Infectious Diseases Research at the College of Veterinary Medicine, North Carolina
State University during 1994 and
1995. He received a PhD in veterinary
parasitology from the Hebrew University in 2000. Prof. Baneth served as
the head of the Small Animal Internal
Medicine Department at the Hebrew
University Veterinary Teaching Hospital. He is a diplomate of the European
College of Veterinary Clinical Pathology (ECVCP) and an editorial advisory
board member for the Journal Veterinary Parasitology since 2006. He is the
vice president of the LeishVet group for
standardization of the diagnosis, treatment and prevention of canine leishmanioasis, a member of the World
Small Animal Veterinary Association
(WSAVA) Scientific Advisory Committee (SAC), member of the International Society for Companion Animal
Infectious Diseases (ISCAID) Board of
Directors, and a member of Board of
Directors, Israel Society for Parasitology, Protozoology and Tropical Diseases. His research interests focus on
the pathogenesis, diagnosis and treatment of veterinary and zoonotic vector-borne infectious diseases including
leishmaniosis, canine ehrlichiosis,
babesiosis, hepatozoonosis, trypanosomiasis and dirofilariasis. Prof.
Baneth is involved in the study of
zoonotic and veterinary diseases in the
Mediterranean Basin, Uzbekistan,
Ethiopia and South America funded by
the European Union and other international research agencies. He is the
author of more than 140 scientific publications and book chapters.
GIOVANNI
BARSOTTI
Med Vet, Dr Ric,
SCMPA, Pisa,
Italy
Giovanni Barsotti received the degree in
Veterinary Medicine cum laude from
Pisa University, Italy, in 1996.
Since 2000 he is an assistant professor in Veterinary Surgery at the Department of Veterinary Sciences of the
University of Pisa, Italy.
Teacher in courses related to animal
surgery – course of Veterinary Medicine, University of Pisa - 2004-2005
to date.
His research interests are in the areas
of veterinary ophthalmology and experimental surgery.
He is author of more than 60 scientific publications, including papers in
peer-reviewed international journals
and two book chapters (“Malattie dell’occhio” in Ailuropatie: dal sintomo
alla terapia, vol.
II di G. Guidi e coll.; “occhio, annessi
oculari e cavità orbitale” in Citologia
del cane e del gatto di A. poli e A.
Ciorba).
He is a member of SISVet (Italian Society of Veterinary Sciences); SICV
(Italian Society of Veterinary Surgery) and SOVI (Italian Society of Veterinary Ophthalmology).
ERIKA CARLI
Med Vet, PhD student,
Padua, Italy
Erika Carli graduated
at Padua University
in 2000. She worked
until 2012 at the San
Marco laboratory in Padua.
Currently, she is a PhD student in veterinary parasitology at Padua University.
She is author of scientific papers in international journals.
She is particularly interested in hematology, transfusional medicine and
vector-borne diseases.
FILIPE
DANTAS-TORRES
DVM, MSc, DSc,
PhD, FRES, Brazil
Dr. Dantas-Torres
graduated in Veterinar y Medicine in
2004 at the Federal Rural University
of Pernambuco in Brazil. In 2006 and
2009 he received a Master of Sciences
and a Doctor of Sciences, respectively, from the Aggeu Magalhães Research Institute.
In 2010, he was elected as Fellow of
the Royal Entomological Society (FRES)
and nominated as Cultore della Materia in Parassitologia e Malattie
Parassitarie. In 2012 he earned the tittle of Dottore di Ricerca from University of Bari, Italy. Dr. Dantas-Torres is
the author or co-author of over 220
publications, including 130 papers in
refereed journals and 5 book chapters.
He is on the editorial board of Medical and Veterinary Entomology and
Parasites & Vectors and reviews for
over 40 journals.
He has lectured extensively throughout Brazil, the USA, Europe, and Asia.
Currently, he is a post-doc research fellow at the Aggeu Magalhães Research Institute, Oswaldo Cruz Foundation, Brazil. He has been involved
in research projects on diagnosis,
epidemiology and control of vectorborne parasitic diseases, including
leishmaniasis and dirofilariosis. He has
extensively studied many aspects related to the brown dog tick (Rhipicephalus sanguineus), the most widespread tick in the world.
LAURA ORDEIX
DVM, Dipl ECVD,
Barcelona, Spain
Laura Ordeix graduated
from the Veterinary
School of Barcelona
(UAB) in 1996. She is
member of the European College of
Veterinary Dermatology since 2002.
She is a co-author of the chapters:
Rigid Endoscopy – Otoendoscopy of
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the Manual of Canine and Feline Endoscopy and Endosurgery (2008,
BSAVA) and Naso e seni paranasali
- Malattie del planum nasale of Malattie dell’apparato respiratorio nel cane
e nel gatto (2012, Elsevier). She has
authored scientific articles in national and international journals. Currently
she is member of scientific committee
of the Annual Congress of the ESVDECVD and chair of dermatology in the
SEVC. Currently she works as a clinical dermatologist in a referral hospital in Barcelona (Ars Veterinaria).
Her areas of special interest are otology, allergology, dermatopathology,
medical education and canine leishmaniosis. She has obtained the ESVD
Major grant 2012 to study on canine
innate immune response against Leishmania infection.
DOMENICO
OTRANTO
Med Vet, Dr Ric,
Dipl EVPC,
FRES, Bari, Italy
He currently is Full Professor of Parasitology
and Animal Parasitic Diseases in the
Faculty of Veterinary Medicine at the
University of Bari, Italy. He is an author
or co-author of more than 250 peerreviewed articles in international scientific journals in the field of parasitology and author of numerous book
chapters in the same field. He is the recipient of the Outstanding Young Scientist award from the World Association for the Advancement of Veterinary
Parasitology (also known as the “Peter Nansen Award”) in 2007 and the
International award of the “Lincei
Academy” for “Evolutionary aspects of
Zoology” in 2006. His research focuses
on the study of arthropods and arthropod borne diseases.
In particular, he has conducted studies
on members of the dipteran family
Oestridae and nematode family The-
laziidae, as well as the diseases they
cause in humans and animals. He currently works on different aspects of canine vector borne diseases of zoonotic concern (e.g., leishmaniasis) with
both a basic and an applied approach. Professor Otranto is Fellow of
the Royal Entomological Society (FRES),
Editor in Chief of Medical and Veterinary Entomology, Advisor Editor of
Parasites and Vectors and editorial
board member of four scientific international journals in the field of the Parasitology.
MARIA GRAZIA
PENNISI
Med Vet, PhD,
Messina, Italy
Maria Grazia Pennisi
qualified from the faculty of Veterinary
Medicine, University
of Messina (Italy) in 1979. She obtained her PhD in applied Microbiology in 1982 and won scholarships to
attend the Istituto Zooprofilattico Sperimentale del Mezzogiorno (Portici,
Naples) and the National Research
Council (Consiglio Nazionale delle
Ricerche, Rome). From 1983 to 1991,
she was appointed researcher in
Clinical Veterinary Medicine at the University of Messina.
In 1991, she became Associate Professor and in 2000 Full Professor in
Clinical Veterinary Medicine at the
same faculty. Since 1997 she is member of the editorial board of the journals “Bollettino AIVPA” and “Rassegna di Medicina Felina”. She was
Board member of the Associazione Italiana Veterinari Patologia Felina (Italian Veterinary Association for Feline
Diseases) from 2005 to 2011 and is
honour member of AIVPA (Associazione Italiana Veterinari Piccoli Animali). Since 2006 she is founding
member of the international association LeishVet.
She has a particular interest in feline
medicine, feline infectious diseases,
clinical immunology and small animal
arthropod borne diseases. She is author or co-author of some 140 papers
published in peer reviewed journals,
book chapters, and is a frequent invited
speaker at national and international
congresses.
XAVIER ROURA
DVM, PhD,
Dipl ECVIM-CA,
Barcelona, Spain
Dr Roura is a veterinarian of the Veterinarian Teaching Hospital at the College of Veterinary Medicine at the Autonomous University of
Barcelona (Spain). Dr. Roura received his
DVM from Autonomous University of
Barcelona (Spain) in 1989.
From 1990 to 1991 he did an internship at the Veterinarian Teaching
Hospital at the College of Veterinary
Medicine at the Autonomous University of Barcelona (Spain).
He received his PhD from Autonomous
University of Barcelona (Spain) in
1999 with the work “Diagnosis of Canine Leishmaniasis with Polymerase
Chain Reaction”.
He became Diplomate in the European
College of Veterinary Internal Medicine
with specialty the Companion Animals
in 2004.
From 1992 until present he has been
working as a clinical instructor at the
Veterinarian Teaching Hospital at the
College of Veterinary Medicine at the
Autonomous University of Barcelona
(Spain).
Dr Roura has been veterinary visitant
of the Veterinary College of Ohio State
(1993) and North Carolina State (1997,
2001 and 2004). He has several scientific publications and book chapters
in the areas of infectious diseases (vector-borne diseases), cardio-respiratory
and immunology.
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COMPANY SPEAKERS
GIADA
BAGNAGATTI
DE GIORGI
Med Vet, Milano
Laureata in Medicina
Veterinaria presso
l’Università degli Studi
di Milano nel 2009. Dal 2010 al
2012 ha frequentato il Dipartimento di
Scienze Veterinarie per la salute, la Produzione Animale e la Sicurezza Alimentare in qualità di dottorando di ricerca svolgendo attività clinica e attività
di ricerca. Dal 2011, dopo un periodo
formativo presso il Centro de Trasfusion
Veterinario di Madrid (Dr. Luis Miguel
Vinals Florez), collabora attivamente
con il Reparto di Medicina Emotrasfusionale Veterinaria (REV) dell’Università
di Milano. Coautrice di 9 pubblicazioni
su riviste nazionali e internazionali. I
suoi principali settori di interesse sono
la medicina interna degli animali da
compagnia, con particolare riguardo
alle malattie infettive (specialmente la
Leishmaniosi canina), e la medicina
emotrasfusionale veterinaria.
LUIGI GRADONI
Scien Biol, Istituto
Superiore di Sanità,
Roma
Studi e formazione:
1982, diploma in “Basic Course on Malaria
and other Tropical Parasite Diseases, Turkey/Italy”; 1981,
stage in Medical entomology, Imperial
College, Ascot, UK; 1978, stage in Fisiologia degli insetti ed Effetto neurotossico del veleno di ragno Latrodectus
tredecimguttatus, Istituto di Fisiologia, Università di Pisa; 1974, stage in Taxonomy of phlebotomine sandflies (Diptera:
Psychodidae), British Museum (N.H.),
London, UK; 1969, laurea in Scienze Biologiche presso La Sapienza Università degli Studi di Roma. Incarichi, attività didattica e di esperto: Direttore del Reparto
di Biologia, sistematica ed ecologia dei
vettori, ISS (1989-2004); Responsabile,
Revisioni di analisi per le contaminazioni
parassitologiche degli alimenti (19932008); Docente ad incarico, corso integrativo della disciplina Zoologia Sistematica: Artropodi d’interesse sanitario,
Università degli Studi della Tuscia, Viterbo
(2000 e 2004); Advisor e consulente per
l’OMS nel campo dei vettori della leishmaniosi in Uzbekistan e Syria. Entomologist expert dell’European Center for
Disease Prevention and Control per la valutazione del rischio trasmissione delle
vector-borne diseases in Malta. TAIEX Entomologist Expert dell’European Commission per il controllo dei vettori di leishmaniosi in Croazia. Principali progetti
scientifici e sintesi delle attività: Progetto EC/LSTM/ ISS “Molecular techniques
for vector and parasite characterization
applied to a pilot vector control study of
leishmaniasis in Venezuela” (19941997). 6° F.R.P. Integrated Project “Emerging Diseases in changing European eNvironment” (EDEN), Subproject Leishmaniasis (EDEN-LEI) (2004-2009).
Le attività di ricerca sono state finalizzate
allo studio degli artropodi d’interesse sanitario e principalmente al ruolo vettore
di agenti patogeni di malattie endemiche in Italia e nei Paesi del bacino del
Mediterraneo. In particolare, le leishmaniosi hanno fornito l'impulso per indagini di campo e di laboratorio sui vettori della parassitosi. Le attività di ricerca all’estero includono Venezuela, Uzbekistan, Cipro, Albania, Croazia e Siria. Pubblicazioni: A marzo 2011 sono
360 i contributi scientifici pubblicati o in
stampa; di questi, 117 sono stati citati da
PubMed.
DAVID MCGAHIE
BVMS, BSc(Vet.Sci.), MRCVS, UK
David graduated from the Glasgow Veterinary School in Scotland in 1998. After 9 years in general practice, latterly with a strong emphasis on companion animal practice, he joined Virbac
UK in 2007 to work as a Veterinary
Adviser. In 2009, he moved to the
South of France to join the Medical Department in Virbac Headquarters, initially as a technical manager with responsibility for the fields of oncology,
immunology and internal medicine. He
was then appointed to the post of Directeur Médical where he is responsible for scientific communication and
phase IV clinical studies at the group level. He has a special interest in immunology and canine leishmaniosis.
MARCO MELOSI
Med Vet, Cecina (LI)
Medico Veterinario, libero professionista di
Cecina (LI), esercita
nel settore della clinica
degli animali da compagnia. Socio SCIVAC di lunga data,
è stato membro della Commissione
ANMVI per la Qualità. È fra gli autori del Manuale ANMVI per le Buone
Pratiche Veterinarie.
Nel mandato precedente ha ricoperto la carica di Vice-Presidente con delega al settore degli animali da compagnia. Attualmente è anche coordinatore dei consigli direttivi delle ANMVI regione.
GAETANO OLIVA
Med Vet, Dr Ric,
Napoli
Si è laureato in Medicina Veterinaria presso l’Università degli
Studi di Napoli Federico II, il 31/07/ 1984. Dal 1984 al
1987 ha trascorso un periodo di formazione presso l’Istituto di Clinica Medica Veterinaria della Facoltà di Napoli. È stato borsista per tre mesi (novembre 1990- febbraio 1991) presso
il Department of Clinical Sciences of
Companion Animals, dell’Università di
Utrecht, Olanda.
Dal 1987 al 1991 è stato Funzionario Tecnico Laureato presso l’Istituto di
Clinica Medica Veterinaria della Facoltà di Med Vet, Napoli. Dal 1991 al
2001 è stato Professore Associato di
Terapia Medica Veterinaria presso la
stessa Facoltà.
Dal 2001 è Professore Ordinario; attualmente ricopre la cattedra di Clinica Medica Veterinaria. Dal 2001 il Prof
Oliva è il Presidente del Corso di Laurea Specialistica in Medicina Veterinaria della Facoltà di Napoli. I suoi interessi didattici e di ricerca sono nel
campo della Medicina Interna degli
animali da Compagnia, con particolare riguardo agli aspetti diagnostici,
clinici e terapeutici delle malattie trasmesse da vettori. È autore di nunmerosi lavori a stampa, su riviste nazionali ed internazionali.
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PROGRAMMA SCIENTIFICO / SCIENTIFIC PROGRAM
VENERDÌ 8 MARZO 2013 / FRIDAY, MARCH 8TH 2013
09.20
Saluto e presentazione del Congresso / Opening cerimony
Chairperson: George Lubas
09.40
Aggiornamenti sull’interazione tra ospite e parassita e lo stato immunitario nella Leishmaniosi canina
What is new on host parasite interaction and immunity in canine Leishmaniosis?
Laura Ordeix (E)
10.20
Quadri clinici comuni ed inusuali nella Leishmaniosi canina (escluse manifestazioni cutanee)
Usual and unusual clinical pictures other than the cutaneous signs associated
with canine Leishmaniosis - Xavier Roura (E)
11.00
Pausa caffè / Coffee break
11.40
Serological and molecular evaluation of Leishmania infantum infection in stray cats in a
non-endemic area in Northern Italy - Giada Bagnagatti De Giorgi (I) (15’)
11.55
Lesioni oculari associate alla Leishmaniosi canina
Ocular diseases associated with canine Leishmaniosis
Giovanni Barsotti (I)
12.35
Coinvolgimento renale in corso di Leishmaniosi canina
Renal involvement in canine Leishmaniasis - Giada Bagnagatti De Giorgi (I)
12.55
Pausa pranzo / Lunch break
Chairperson: Alessandra Fondati
14.10
Lesioni cutanee inusuali associate alla Leishmaniosi canina
Unusual cutaneous diseases associated with canine Leishmaniosis - Laura Ordeix (E)
14.50
Confronto tra indagini diagnostiche invasive e non invasive nella Leishmaniosi canina
A comparison of non-invasive and invasive diagnostic methods for canine Leishmaniosis
Gad Baneth (IL)
15.30
Retrieval of leishmanial DNA in a commercially available LiESP/QA-21 vaccine
Tommaso Furlanello (I) (15’)
15.45
Strategie tradizionali e innovative nella terapia della Leishmaniosi
Old and New strategies of Leishmaniosis treatment - Xavier Roura (E)
16.25
17.05
Misdiagnosi di linfoma in un cane con Leishmaniosi
Fabrizio Ibba (I) (15’)
17.20
CVBD Clinical Center: un supporto al Clinico per la gestione delle Malattie Trasmesse da Vettore
CVBD Clinical Center: A support to the practitioner for vector born disease management - Gaetano Oliva (I)
17.40
Nuove strategie nella prevenzione della Leishmaniosi
New strategies of Leishmaniosis prevention - Xavier Roura (E)
18.20
Termine della giornata / End of the day
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SABATO 9 MARZO 2013 / SATURDAY MARCH 9th 2013
Chairperson: Gad Baneth
09.40
Leishmaniosi felina: tra certezze e dubbi
Update on feline Leishmaniosis - Maria Grazia Pennisi (I)
10.20
Epidemiology, seroprevalence and management of canine visceral Leishmaniasis in Paraguay
by the national programme of zoonoses control - Jorge Miret (PY) (15’)
10.35
11.15
Coinfezioni tra Leishmaniosi e altre malattie del cane trasmesse da vettori
Leishmaniosis and co-infections with other canine vector-borne diseases
Xavier Roura (E)
11.55
Rapid stimulatory effect of Leisguard® on the canine innate immune system - David Sabatè (E) (15’)
12.10
Nuova epidemiologia delle malattie trasmesse da vettori
Changing epidemiology of vector-borne diseases - Filipe Dantas-Torres (BR)
12.50
SCALIBOR Map Update: Mappatura Nazionale zone a rischio Leishmaniosi 2013
Scalibor Map Update: National Mapping of areas at risk Leishmaniasis 2013 - L. Gradoni (I) - M. Melosi (I)
13.10
Pausa pranzo / Lunch break
Chairperson: Laura Ordeix
14.20
Ehrlichiosi canina
Canine ehrlichiosis - Gad Baneth (IL)
15.00
Serological and clinical evidence of Leishmaniosis in a dog population living in a farm
in Northern Serbia - Sara Savic (SRB) (15’)
15.15
Fisiopatologia e terapia della Babesiosi. Nuove forme di Piroplasmosi nel cane e nel gatto
Pathophysiology and treatment of Babesiosis. Emergence of new piroplasms/diseases in dogs
and cats - Gad Baneth (IL)
15.55
16.40
Micoplasmosi
Mycoplasmosis - Xavier Roura (E)
17.20
Mycoplasma haemofelis and ‘candidatus mycoplasma haemominutum’ distribution in distinct feline
population, Portugal - Cátia Marques (15’)
Nuove malattie trasmesse da vettori nel Vecchio e nel Nuovo Mondo
New canine vector-borne diseases in the Old and in the New World Domenico Otranto (I) e Filipe Dantas-Torres (BR)
La Leishmaniosi e il cane, una partita tutta da giocare
Immunopatogenesi della Lcan per clinici - Xavier Roura (E)
Leishvet clinical staging: inquadrare bene per colpire meglio - Gaetano Oliva (I)
Canileish, new data from the field - David McGahie (UK)
Dog and Leishmaniosis: a game to play from the beggining
Lcan immunopathogenesis for clinicians - Xavier Roura
Leishvet clinical staging: framing well, to hit better - Gaetano Oliva
Canileish, new data from the field - David McGahie
17.35
18.15
18.35
Termine della giornata / End of the day
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DOMENICA 10 MARZO 2013 / SUNDAY MARCH 10th 2013
Chairperson: Walter Bertazzolo
09.00
Che cosa sappiamo del Cytauxzoon in Europa?
What is known about Cytauxzoon in Europe? - Erika Carli (I)
09.40
Haematological profiles in canine monocytic ehrlichiosis: a retrospective study of 31 spontaneous cases
in Greece - Ilia Tsachev (BG) (15’)
09.55
Aggiornamenti sulla Epatozoonosi canina e felina
What we really know about canine and feline Hepatozoonosis - Gad Baneth (IL)
10.35
11.20
Malattie canine trasmesse da vettori a rischio di zoonosi
Canine vector borne disease of human concern - Domenico Otranto (I)
12.00
Anaplasma platys, Ehrlichia canis and Leishmania infantum infection in a Sardinian dog infested
by ticks - Antonio Viglietti (I) (15’)
12.15
Discussione di casi clinici di malattie trasmesse da vettori: fai la tua diagnosi
Clinical cases of vector borne diseases discussed between speakers and delegates:
make your diagnosis! - Walter Bertazzolo
12.55
Termine del Congresso / End of the Congress
POSTERS
MOLECULAR SURVEY OF LEISHMANIA, EHRLICHIA, ANAPLASMA AND BABESIA
GENUS IN WOLVES [CANIS LUPUS SIGNATUS] FROM NORTHEN PORTUGAL
Ana Duarte Lisbona, Portogallo
LEISHMANIA INFANTUM PCR POSITIVE LYMPH NODE ASPIRATES:
CYTOLOGIC PATTERNS IN CATS
Maria Grazia Pennisi, Messina, Italia
FLAVIVIRAL CENTRAL EUROPEAN TICK-BORNE MENINGOENCEPHALITIS:
NATURAL HISTORY AND CLINICO-PATHOLOGICAL FEATURES OF 37 CASES
PRESENTED BETWEEN 1999 - 2012
Lorenzo Golini, Zurigo, Svizzera
STUDIO CLINICO RETROSPETTIVO SULLA PREVALENZA SIEROLOGICA DELLE
MALATTIE EMOPROTOZOARIE IN UNA POPOLAZIONE CANINA
Stefania Pitarra, Fragagnano (TA), Italia
HAEMATOLOGICAL CHANGES IN DOGS DURING THE APPROPRIATE SEASON
OF THE YEAR AS AN INDICATOR FOR ACUTE CANINE BABESIOSIS
Geroges Kirtz, Vienna, Austria
ANAPLASMOSIS IN DOGS IN AUSTRIA: A SEROPREVALENCE STUDY
Geroges Kirtz, Vienna, Austria
USEFULNESS OF A SEROLOGICAL SIMULTANEOUS DIAGNOSIS OF FOUR CANINE
VECTOR-BORNE DISEASES IN DOGS - Maria Renzi, Bologna, Italia
CANINE LEISHMANIASIS AND BIOLOGICAL NETWORKS:
A NEW APPROACH FOR AN OLD PROBLEM
Daniela Rispoli, Mosciano Sant'angelo (TE), Italia
RETROSPECTIVE STUDY OF CANINE EHRLICHIOSIS IN BUENOS AIRES
Ilaria Lippi (UK)
EVALUATION OF THE HUMORAL IMMUNE RESPONSE AFTER THE FIRST
ANNUAL CANILEISH® BOOSTER VACCINATION
Emmanuelle Sagols, La Seyne Sur Mer, France
CANINE LEISHMANIASIS: A COMPARATIVE STUDY OF REAL-TIME PCR IN
CONJUCTIVAL AND WHOLE BLOOD SAMPLES AND INDIRECT FLUORESCENT
ANTIBODY TEST FOR THE DETECTION OF LEISHMANIA INFANTUM
Afroditi Mountousi, Atene, Grecia
AUTOCHTHONOUS CASES OF DIROFILARIA REPENS IN GERMANY,
AN EXAMPLE FOR THE SPREADING OF A VECTOR BORNE DISEASE AS
A RESULT OF CLIMATE CHANGE
Reinhold Sassnau, Berlino, Germania
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ESTRATTI DELLE RELAZIONI
Questo volume di atti congressuali riporta fedelmente quanto fornito dagli autori
che si assumono la responsabilità dei contenuti dei propri scritti.
Gli estratti sono elencati in ordine alfabetico secondo il cognome del relatore
e quindi in ordine cronologico di presentazione.
These proceedings report faithfully all abstracts provided by the authors
who are responsible of the content of their works.
The abstracts are listed in alphabetical order by surname
and then in chronological order of presentation.
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Gad Baneth
DVM, PhD, Dipl ECVCP
Israel, Jerusalem
A comparison of non-invasive
and invasive diagnostic methods
for canine leishmaniosis
Friday, March 8th 2013 - 2.50 pm
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Canine leishmaniosis (CanL) is a major zoonotic disease endemic in more
than 70 countries in the world. It is enzootic in regions of southern Europe,
Africa, Asia, South and Central America and has recently emerged in the
USA. CanL is also an important concern in non-endemic countries where imported disease constitutes a veterinary and public health problem. Dogs are
the main animal reservoir for human visceral leishmaniasis and the disease is
usually fatal if not treated in people. Seroprevalence rates found in studies carried out in the Mediterranean basin range between 10 and 37% of the dogs in
disease foci. Surveys employing the polymerase chain reaction (PCR) for the
detection of leishmanial DNA in canine tissues, or combining serology and
DNA detection, have revealed even higher infection rates approaching 70% in
some foci. It has been estimated based on seroprevalence studies from Italy,
Spain, France and Portugal that 2.5 million dogs in these countries are infected. The number of infected dogs in South America is also estimated in millions with high infection rates in some areas of Brazil and Venezuela1,2.
THE PATHOGENESIS OF CANINE LEISHMANIOSIS
The concept that all dogs infected with L. infantum will eventually develop severe clinical leishmaniosis after a variable incubation period has been
disproven and the classical stages of an infectious disease process which includes an initial infection, an incubation period and a clinical disease apply
only in the minority of dogs with Leishmania infection. Leishmania infantum
infection can elicit a broad spectrum of immune responses and display a variety of clinical manifestations in dogs ranging from a clinically healthy animal to a severe clinical disease condition. The two opposite extreme poles of
this spectrum are characterized by protective immunity that is T cell mediated, or disease susceptibility associated with the production of a marked humoral non-protective immune response and a reduced or depressed cell mediated immunity.
Population studies in Leishmania-endemic areas have shown that a proportion of the canine population develops a clinical disease, another fraction
has persistent asymptomatic infection, while yet another fraction is resistant
to the infection or intermittently resolves it without developing clinical
signs1,2,3. The immune responses mounted by dogs at the time of infection and
thereafter appear to be an important factor in determining if they will develop
a lasting infection and whether and when it will progress from an asymptomatic state into a symptomatic disease. Dogs that are able to resist infection
and either resolve it and eliminate the parasite, or restrict the infection and remain constantly asymptomatic, have been termed “clinically resistant”. Ani17
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mals that are predisposed and will develop symptomatic disease are considered “susceptible”.
DIAGNOSIS
CanL is a good example of a disease in which infection does not equal clinical illness due to the high prevalence of asymptomatic infection. This makes
CanL a diagnostic challenge for the veterinary practitioner, clinical pathologist
and public health official in endemic countries as well as non-endemic regions
where imported infection is a concern2,4,5,6. Accurate diagnosis of CanL often
requires an integrated approach (clinicopathological diagnosis and specific
laboratory tests) which includes careful documentation of the clinical history,
a thorough physical examination and several diagnostic tests such as CBC, biochemical profile, urinalysis, urine protein/creatinine ratio, serum electrophoresis, and a coagulation profile. Imaging of the abdomen by radiographs and ultrasound can assist in raising the suspicion index for this disease.
SPECIFIC DIAGNOSIS
The most useful diagnostic approaches for investigation of infection in
sick and clinically healthy infected dogs include detection of serum anti-leishmanial antibodies by a quantitative serological assay and demonstration of the
parasite DNA in tissues by applying molecular techniques.
Serology: Various serological methods for the detection of anti-Leishmania antibodies have been developed. These include indirect immunofluoresence assays (IFA), enzyme-linked immunosorbent assay (ELISA), direct agglutination assays (DAT) and western blotting. A purified recombinant antigen for ELISA, rK39, has been used for detection of visceral leishmaniosis in
humans and dogs. In general, good sensitivities and specificities are gained
with quantitative serological methods for the diagnosis of clinical CanL. High
antibody titers are usually associated with disease and a high parasite density
and for this reason they are conclusive of a diagnosis of leishmaniosis. However, the presence of lower antibody levels is not necessarily indicative of
patent disease and needs to be confirmed by other diagnostic method such as
PCR, cytology or histology. Serological cross-reactivity with different
pathogens is possible with some serological tests, especially those based on
whole parasite antigen. Cross reactivity has been reported with other species
of Leishmania and Trypanosoma cruzi.
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Cytology: Leishmania amastigotes can be demonstrated by cytology from
lymph nodes, spleen or bone marrow stained with Giemsa stain or a quick commercial stain. Detection of amastigotes by cytology is frequently unrewarding
due to a low number of detectable parasites present even in dogs with a full
blown clinical disease. Leishmania amastigotes may also be visualized in
histophatologic formalin-fixed, paraffin-embedded biopsy sections of the skin
or other infected organs. Definite identification of amastigotes within tissue
macrophages may be difficult and an immunohistichemical staining method
can be employed to detect or verify the presence of Leishmania in the tissue.
PCR: Several PCR assays with various target sequences using genomic or
kinetoplast DNA (kDNA) have been developed for CanL. Assays based on
kDNA appear to be the most sensitive for direct detection in infected tissues.
PCR can be performed on DNA extracted from tissues, blood, biological fluids or from histopathologic specimens. PCR on bone marrow, lymph node,
spleen or skin is most sensitive and specific for the diagnosis of CanL while
PCR on whole blood, buffy coat, and urine is less sensitive than the aforementioned tissues. PCR on aspirates of lymph node and bone marrow has
been shown to be more sensitive than microscopic detection of amastigotes in
stained smears or parasite culture. Quantitative real-time PCR can detect extremely low parasitic loads and allows the quantification of Leishmania loads
in tissues of infected dogs which is important for diagnosis as well as for follow-up during the treatment of CanL.
PCR is not the first confirmatory assay recommended for dogs with clinical signs suspected of CanL because in endemic areas, a large portion of the
dog population is likely to harbor Leishmania without a clinical disease, or
while suffering from a different medical condition. Since high serological titers
are closely associated with clinical disease and less frequent among clinically
healthy carriers of Leishmania, quantitative serology would be the first recommended specific assay for the disease. The presence of Leishmania DNA in the
blood or other tissues of clinically healthy dogs living in endemic areas indicates that these dogs harbor infection, but they may never develop clinical disease. The interpretation of PCR results should be done cautiously in clinically
healthy dogs and with consideration of the diagnostic procedure’s purpose. For
instance, for the purpose of identifying infected dogs and preventing their importation to non-endemic areas where infection might spread via local sand fly
vectors, or for the purpose of preventing transmission of infection via blood
products from infected donors, PCR would be an appropriate technique in
combination with quantitative serological tests. However, the decision to treat
clinically healthy dogs in endemic areas with anti-leishmanial medication
based on positive PCR alone is not recommended.
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NON-INVASIVE DIAGNOSTIC TECHNIQUES FOR CANINE
L. INFANTUM INFECTION
“Non-invasive” diagnostic techniques would consist of sampling techniques that do not require penetration of the skin or a mucosal barrier in order to obtain the sample material. In that respect, venipuncture to obtain
blood; aspiration of the bone marrow, lymph node or spleen; and skin biopsies or scraping, are invasive sampling techniques3,4,6. Conjunctival and oral
mucosal swabs, saliva analysis, PCR on hair, and urine tests using free catch
urine, are non-invasive techniques7-14.
Non-invasive conjunctival PCR has been shown to be accurate in the diagnosis of seropositive dogs with clinical leishmanioasis direct diagnosis of
canine infection7,8, and also useful in the detection of asymptomatic infection9,10. Oral swab PCR has been shown to be considerably less sensitive than
conjunctival PCR11. Urine PCR may also be useful in the detection of infection12,13, and recently PCR of hair has been shown to be sensitive for detection
of infection in dogs with clinical disease14.
It appears that non-invasive conjunctival PCR is a good molecular tool for
the detection of infection in epidemiological surveys’ clinical studies, and in
clinical situations when invasive detection of infection is not desirable.
REFERENCES
1. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. 2008. Canine leishmaniosis- New Concepts and Insights on an Expanding Zoonosis – part one. Trends Parasitol. 24:324-330.
2. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P,
Oliva G, Baneth G.Directions for the diagnosis, clinical staging, treatment and prevention
of canine leishmaniosis. Vet. Parasitol. 165: 1-18.
3. Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L. 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity
using PCR on several tissues and serology. J. Clin. Microbiol. 39: 560-563.
4. Baneth and Aroch. 2008. Canine leishmaniasis – a diagnostic and clinical challenge. Vet.
J. 175:14-15; 5.
5. Miró G, Cardoso L, Pennisi MG, Oliva G, Baneth G. 2008. Canine leishmaniosis- New
Concepts and Insights on an Expanding Zoonosis – part two. Trends Parasitol. 24: 371-377.
6. Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P,
Oliva G, Baneth G. 2011. The LeishVet Group. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 4:86.
7. Strauss-Ayali D, Jaffe CL, Burshtain O, Gonen L, Baneth G. 2004.Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum
DNA in dogs. J Infect Dis. 189:1729-1733.
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8. Ferreira Sde A, Ituassu LT, de Melo MN, de Andrade AS. 2008. Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCR-hybridization in Minas Gerais State, Brazil. Vet Parasitol. 152:257-263.
9. Leite RS, Ferreira Sde A, Ituassu LT, de Melo MN, de Andrade AS. 2010. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples. Vet
Parasitol. 170:201-206.
10. Di Muccio T, Veronesi F, Antognoni MT, Onofri A, Piergili Fioretti D, Gramiccia M.
2012. Diagnostic value of conjunctival swab sampling associated with nested PCR for
different categories of dogs naturally exposed to Leishmania infantum infection. J Clin
Microbiol. 2012 50:2651-2659.
11. Lombardo G, Pennisi MG, Lupo T, Migliazzo A, Caprì A, Solano-Gallego L., 2012. Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival
swabs and comparison with other diagnostic techniques. Vet Parasitol. 184:10-17.
12. Belinchón-Lorenzo S, Iniesta V, Parejo JC, Fernández-Cotrina J, Muñoz-Madrid R, Soto M, Alonso C, Gómez Nieto LC. 2013. Detection of Leishmania infantum kinetoplast
minicircle DNA by Real Time PCR in hair of dogs with leishmaniosis. Vet Parasitol.
192:43-50.
13. Solano-Gallego L, Rodriguez-Cortes A, Trotta M, Zampieron C, Razia L, Furlanello T,
Caldin M, Roura X, Alberola J. 2007. Detection of Leishmania infantum DNAby fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol.
147:315-319.
14. Manna L, Reale S, Picillo E, Vitale F, Gravino AE. 2008. Urine sampling for real-time
polymerase chain reaction based diagnosis of canine leishmaniasis. J Vet Diagn Invest.
20:64-27.
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Gad Baneth
Jerusalem, Israel
Canine ehrlichiosis
Saturday, March 9th 2013 - 2.20 pm
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Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, has
been recognized worldwide as an important canine infectious agent (Harrus
and Waner 2011). It has been reported from Africa, Asia, America, and Europe. Cases of E. canis in Europe have been reported mostly from Spain, Portugal, Southern France, Italy, the Balkans, Turkey and Greece.
Ehrlichia canis is transmitted by the three-host tick Rhipicephalus sanguineus. The pathogenesis of the disease involves an incubation period of 820 days, followed by 3 consecutive phases: an acute phase which lasts 1-4
weeks, a subclinical phase which may last from months to years, and a chronic phase. Not all infected dogs develop the chronic severe form of the disease
and the conditions that lead to the development of this stage are unknown
(Cohn 2003; Harrus and Waner 2011). Ehrlichia canis can also be transmitted by blood transfusion and it is recommended to screen for its presence in
the blood of donor dogs.
CLINICAL FINDINGS
The clinical presentation of the disease caused by E. canis may vary (Harrus et al., 1997; Frank and Breitschwerdt 1999; Cohn 2003; Gaunt et al.,
2010; Harrus and Waner 2011), and the clinical signs most frequently reported are lethargy, anorexia, fever, lymphadenomegaly, splenomegaly and hemorrhages, mainly petechiae, ecchymoses and epistaxis. Ocular manifestations
of canine ehrlichisosis include anterior uveitis, keratoconjuctivitis, hyphema,
glaucoma, chorioretinitis and retinal detachment.
Polyarthritis and polymyositis have also been described in E. canis infection. The neurological abnormalities found in canine ehrlichiosis are associated with vasculitis, meningoencephalitis, and lymphocytic infiltration of the
central and peripheral nervous system or hemorrhages. Ehrlichia canis infection has been termed by some clinicians as the “silent killer”. It is often not
apparent during the early and sub-clinical stages of infection, and when the
disease is diagnosed in the chronic stage, it may be too late to save the canine
patient, as treatment may not be helpful in reversing the severe pancytopenia
caused by bone marrow suppression associated with this disease.
Laboratory abnormalities in canine monocytic enrlichiosis include hematologic and serum biochemistry changes (Harrus et al., 1997; Frank and Breitschwerdt 1999; Cohn 2003; Harrus and Waner 2011). Thrombocytopenia
is the most frequent hematological abnormality occurring in more than 90%
of cases. Anemia, usually non-regenerative normocytic and normochromic, is another common finding in this disease. In addition, mild to severe leucopenia
is a frequent abnormality. Hyperglobulinemia, hypoalbuminemia and mild
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elevation of alkaline phosphatase (ALP) and alanine aminotransferase
(ALT) activities are frequently reported in ehrlichiosis. Dogs in the chronic
severe stage of the disease may develop severe pancytopenia as their bone
marrow becomes hypocellular. The prognosis of these chronically ill dogs
is grave.
Immune-mediated responses play a major role in the pathogenesis of E.
canis infection. Anti-platelets antibodies have been demonstrated less than a
week after experimental E. canis infection of dogs. Platelet aggregation abnormalities, anti-nuclear antibodies, RBC autoagglutination with positive
coombs’ test, and circulating immune-complexes have been shown in infected dogs and are associated with the disease process (Harrus and Waner 2011).
The decrease in platelets during canine ehrlichiosis is a result of several
mechanisms. These mechanisms include increased consumption with vascular endothelial changes, platelet sequestration and pooling in the spleen,
thrombophagocytosis with immunological destruction, a decrease in the
half life time of circulating platelets possibly due to opsonization with antibodies, and production impairment due to bone marrow destruction and
hypocellularity. In addition to the decrease in circulating platelet number,
platelets dysfunction (thrombocytopathy) has also been implicated as an additional factor contributing to lack of platelet functionality in canine monocytic ehrlichiosis.
DIAGNOSIS
The laboratory diagnosis of E. canis infection includes evaluation of the
hemogram and serum biochemistry panel (Cohn 2003; Harrus and Waner
2011).
Cytology: Ehrlichia canis morulae found in monocytes and macrophages
are a “microcolony” of bacteria surrounded by a membranous vacuole. Morulae may contain 100 or more ehrlichiae organisms. The detection of morulae
in monocytes in stained blood smears is rare and cannot serve as a main diagnostic option.
PCR: Detection of the presence of E. canis DNA by PCR is highly sensitive and specific and has become the most useful diagnostic test for the confirmation of canine ehrlichiosis. Several conventional and real-time PCR protocols have been described for E. canis and the assay can be performed on
blood or tissue including the spleen and bone marrow (Baneth et al., 2009;
Gaunt et al., 2010; Harrus and Waner 2011).
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Serology: Anti-E. canis antibodies persist long after recovery from the disease. Serum antibodies are thought not to be protective or play an important
role in eliminating this intracellular infection. Serology is indicative of exposure to E. canis and may often be helpful in ruling out progressive infection.
Antibodies may not be detectable during the early stage of infection. However, seropositive dogs with previous exposure to the pathogen may also present
due to other urgent disease conditions.
TREATMENT
Ehrlichis canis is susceptible to doxycycline which is highly efficient in
clearing rickettsemia in acute cases of E. canis infection (Cohn 2003). Clinical recovery is noticed within 48-72 hours, yet treatment should be continued
for 3 weeks, as some dogs may remain carriers when shorter treatments are
applied. Treatment with the injectable drug imidocarb dipropionate has been
shown to be ineffective in totally eliminating E. canis. However, it is often
used in combination with doxycyline when Babesia co-infection is suspected.
PREVENTION
The control of tick infestation by topical treatment with acaricidals and environmental eradication of ticks is recommended for the prevention of E. canis infection. No commercial vaccine against E. canis infection is currently
available.
ANAPLASMA PLATYS
Anaplasma platys was first identified in 1978 in Florida (Harvey et al.,
1978). It is a Gram-negative, obligate intracellular bacterium belonging to the
family Anaplasmataceae and closely related to Anaplasma phagocytophilum
and Ananplasma marginale. Anaplasma platys infects canine platelets and
causes a disease commonly recognized as infectious canine cyclic thrombocytopenia. The presumed natural vector of A. platys is the tick R. sanguineus
and DNA of this bacterium has been reported from this tick species in several countries, however, experimental infection studies have not demonstrated
transmission by R. sanguineus conclusively (Simpson et al., 1991; Gaunt et
al., 2010). Like E. canis, A. platys may also be accidentally transmitted by
blood transfusion (Simpson et al., 1991).
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CLINICAL FINDINGS
A. platys causes a cyclic thrombocytopenia that could result in bleeding,
including petechiae and ecchymoses, although most infected dogs are probably able to control infection without demonstrating clinical signs.
Bacteremia and thrombocytopenia occur in cycles of approximately 10 to
14 days. The clinical findings associated with infection according to published studies include: anorexia, lethargy, fever, weight loss, lymphadenomegaly, petechiae and ecchymoses, thrombocytopenia and anemia (Kontos
et al., 1991; Harrus et al., 1997).
Other studies have described asymptomatic natural infection associated
with A. platys. Infected dogs are frequently co-infected with E. canis, and an
experimental infection of dogs with both agents has shown that dogs with simultaneous infections had more severe clinical manifestations than those infected only with one agent (Gaunt et al., 2010).
DIAGNOSIS
Detection of A. platys morulae in canine platelets can be made upon examination of Giemsa-stained blood smear by microscopy, however, confirmation of infection should be made by specific PCR.
TREATMENT
Anaplasma platys is susceptible to doxycycline with at the same dose and
duration used for E. canis treatment (Gaunt et al., 2010).
REFERENCES
Baneth, G., Harrus, S., Ohnona, F.S., Schlesinger, Y., 2009. Longitudinal quantification of
Ehrlichia canis in experimental infection with comparison to natural infection. Vet. Micro. 136, 321-325.
Cohn, 2003. Ehrlichiosis and related infections. Vet. Clin. North Am. Sm. Animal Prac. 33,
863-884.
Frank, JR., Breitschwerdt, E.B., 1999. A retrospective study of ehrlichiosis in 62 dogs from
North Carolina and Virginia. J. Vet. Intern. Med. 13, 194-201.
Gaunt, S., Beall, M., Stillman, B., Lorentzen, L., Diniz, P., Chandrashekar, R., Breitschwerdt,
E., 2010. Experimental infection and co-infection of dogs with Anaplasma platys and
Ehrlichia canis: hematologic, serologic and molecular findings. Parasit. Vectors. 3, 33.
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Harrus, S., Kass, P.H., Klement, E., Waner, T.l., 1997. Canine monocytic ehrlichiosis: a retropsective study of 100 cases, and an epidemiological investigation of prognostic indicators for the disease. Vet. Rec. 141, 360-363.
Harrus, S., Waner, T., 2011, Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis):
an overview. Vet. J. 187, 292-296.
Gaunt, S., Beall, M., Stillman, B., Lorentzen, L., Diniz, P., Chandrashekar, R., Breitschwerdt,
E., 2010. Experimental infection and co-infection of dogs with Anaplasma platys and
Ehrlichia canis: hematologic, serologic and molecular findings. Parasit. Vectors. 3, 33.
Harrus, S., Aroch, I., Lavy, E., Bark, H., 1997. Clinical manifestations of infectious canine cyclic thrombocytopenia. Vet Rec. 141, 247-250.
Harvey, J.W., Simpson, C.F., Gaskin, J.M., 1978. Cyclic thrombocytopenia induced by a Rickettsia-like agent in dogs. J. Infect. Dis. 137, 182-188.
Kontos, V.I., Papadopoulos, O., French, T.W., 1991. Natural and experimental canine infections
with a Greek strain of Ehrlichia platys. Vet. Clin. Pathol. 20, 101-105.
Simpson, R.M., Gaunt, S.D., Hair, J.A., Kocan, K.M., Henk, W.G., Casey, H.W., 1991. Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys.
Am. J. Vet. Res. 1991 52, 1537-1541.
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Gad Baneth
Jerusalem, Israel
Pathophysiology and treatment
of Babesiosis. Emergence
of new piroplasms/diseases
in dogs and cats
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Babesiosis is a tick-borne protozoal disease with a worldwide distribution
and global importance. Canine babesiosis was reported from South Africa in
1893 and the first record of canine Babesia infection in Europe was made in
Italy in 1895 not long after the detection of bovine babesiosis in Rumania by
Babes in 1888 (reviewed by Roncalli Amici 2001; Uilenberg 2006; and Penzhorn 2011). Babesia infection in dogs was identified in the past based on the
morphologic appearance of the parasite in erythrocytes.
All large forms of Babesia (2.5–5.0 m) were designated Babesia canis,
whereas all the small forms (1.0–2.5 m) were considered as Babesia gibsoni
(Boozer and Macintire, 2003). However, the development of molecular methods have demonstrated that more piroplasmid species infect dogs and cause
different diseases. Babesia rossi, B. canis and B. vogeli previously considered
as subspecies are identical morphologically but differ in the severity of clinical manifestations which they cause, their tick vectors, genetic characteristics,
and geographic distributions, and are therefore currently considered separate
species. Another yet unnamed large Babesia sp. most closely related to B.
bigemina was found to infect immunocompromised dogs in North America
(Birkeneheuer et al., 2004a; Sikorski et al., 2010). The small Babesia spp, include B. gibsoni, B. conradae described from California), and the B. microtilike piroplasm also referred to as Theileria annae or the “Spanish dog isolate”
prevalent in Galicia in north-western Spain (Zhaler et al., 2000; CamachoGarcia et al., 2006).
It is likely that Babesia spp.of dogs originated from wild animals such as
wild canids and infected domestic dogs (Canis familiaris) upon contact with
these adapted reservoir hosts or their ticks. Babesia rossi was first described
from a side-striped jackal (Canis adustus) and later in a jackal pup (reviewed
by Penzhorn 2011). Infection with B. rossi was also prevalent in African wild
dogs (Lycaon pictus) in South Africa. Considering the facts that these wild
canids do not usually suffer from clinical infection with babesiosis and that
the domestic dog was not indigenous to Africa, but rather introduced into subSaharan Africa, it is likely that domestic dogs acquired babesial infection in
Africa from other non-domesticated animals (Penzhorn 2010). Similarly,
Babesia microti-like infections have been reported in foxes in Spain, Portugal
and North America which constitute a reservoir for this infection.
The geographical distribution of the causative agents and thus the occurrence of babesiosis are largely dependent on the habitat of relevant tick vector species, with the exception of B. gibsoni where evidence for dog to dog
transmission indicates that infection can be transmitted among fighting dogs
breeds independently of the limitations of vector tick infestation. Babesia vogeli and B. gibsoni have wide distributions in both the Old and New World
continents, whereas B. rossi has to date been mostly restricted to Africa and
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B. canis has mostly been reported from Europe. Several species of Babesia
have been described in dogs in Europe. Molecular studies on canine Babesia
infection in Europe have reported B. canis infection in Croatia, Poland, Holland, Hungary, Russia, Switzerland, Turkey, Norway and Germany. Infections
with both B. canis and B. vogeli have been reported in Slovenia, France,
Spain, Portugal, Italy and Albania. Babesia microti-like piroplasm infections
were reported in north-western Spain, Portugal, and Coratia. Occasional clinical cases of B. gibsoni were reported in Spain, Germany and Italy (SolanoGallego and Baneth, 2011).
LIFE CYCLE AND TRANSMISSION
Dogs are infected when Babesia sporozoites are injected with saliva into
the host’s skin during the blood meal. The parasites invade the erythrocytes
and form ring-shaped trophozoites. The parasite replicates within the erythrocyte and forms merozoites observed as pairs of attached pear-shaped parasites
in some Babesia species. Merozoites may further divide forming 8 or more
parasites in the same erythrocyte and eventually destroying the cell freeing into the blood to invade more erythrocytes. Ticks feeding on infected blood take
up merozoites and sexual parasite development in the tick gut is followed by
sporogony in its tissues. The parasite reaches the tick salivary glands or it’s
oocytes from which transmission occurs. Babesia spp. are transmitted
transstadially from one stage in the tick life cycle to another, and also transovarially through the tick eggs, as shown for some Babesia spp, The transmission of babesiae occurs through the bite of a vector tick. However, B. gibsoni
infection has also been demonstrated to be transmitted via blood transfusion
and transplacentally. Furthermore, several studies have provided evidence that
B. gibsoni is likely transmitted directly from dog to dog via bite wounds, saliva, or ingested blood (Birkenheuer et al., 2005; Yeagley et al., 2009).
PATHOGENESIS AND CLINICAL MANIFESTATIONS
Hemolytic anemia and the systemic inflammatory response syndrome
leading to multiple-organ dysfunction syndrome are responsible for most of
the clinical signs observed in canine babesiosis. Hemolysis may result in hemoglobinemia, hemoglobinuria, bilirubinemia and bilirubinuria. Thrombocytopenia is observed in many cases of babesiosis and may be caused by immune
mechanisms, splenic sequestration or coagulatory consumption of platelets
from hemolytic or vascular injury. Immune mediated thrombocytopenia has
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been demonstrated in experimental canine babesiosis caused by B. gibsoni.
Tissue hypoxia is found in severe canine bebesiosis. It is caused by anemia, hypotensive shock, vascular stasis by sludging of erythrocytes, excessive
endogenous production of carbon monoxide, and parasitic damage to hemoglobin (Ayoob et al., 2010a). The central nervous system, kidney, and muscle
are the organs most affected by tissue hypoxia (Jacobson, 2006). Hypoxia is
thought to be more important than hemoglobinuria in damaging the kidneys
of dogs with babesiosis (Ayoob et al., 2010a). Tissue hypoxia, hypertensive
shock, multiple organ dysfunction and potential mortality have been documented mostly in association with B. rossi infection. The clinical manifestations of infection with other large Babesia spp. are usually less severe and
typically vary from a mild to moderate disease with B. vogeli to a moderate
disease with B. canis. Young pups and immunocompromised adult dogs, such
as dogs with hyperadrenocorticism or treated with immunsupressive therapy,
may suffer a severe disease with B. vogeli infection.
The spleen has an important function in controlling babesiosis. Experimentally infected splenectomized dogs rapidly develop parasitaemia and clinical disease and may reach high parasitaemia levels. Splenectomy has also
been associated with natural canine and human babesiosis.
DIAGNOSIS
Detection of Babesia in stained blood smears has been the standard diagnostic technique for many years. This method is reliable when a moderate to
high parasitaemia is present. However, a direct correlation between the level
of Babesia parasitaemia and the magnitude of clinical signs is not always
found (Irwin 2010). A fresh smear is recommended for the accurate diagnosis of infection. Erythrophagocytosis with infected erythrocytes may be found
in blood smears from infected dogs. The use of molecular diagnostic assays
is indicative in cases of low parasitemia including suspected carrier dogs or
chronically infected animals.
TREATMENT
Large Babesia spp. are commonly treated with imidocarb dipropionate
with good clinical response while small Babesia spp. appear to be more difficult to treat and resistant to the conventional drugs that are effective against
the large babesial spp.. Diminazene aceturate used for treatment of both large
and small babesial spp. infections should be used cautiously as it has a rela31
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tively small dose safety margin with a large inter-individual pharmacokinetic
variation. Babesia gibsoni infection is often resistant to imidocarb dipropionate and diminazene aceturate and an alternative therapy with the combination of the anti-malarial atovaquone and the macrolide azithromycin has been
recommended for this infection (Birkenheuer et al., 2004b). However, clinical
and parasitological cure are commonly not achieved in small babesial spp. infections and clinical relapses may occur frequently (Irwin 2010). Medical
management of infection may require supportive treatments including blood
transfusions, intravenous fluids, and the use of anti inflammatory drugs (Ayoob et al., 2010a; Irwin 2010).
PREVENTION
Prevention of babesiosis relies mostly on topical and environmental acaricidal treatments aimed at reducing the exposure to vector ticks and the transmission of the pathogen to the dog. Decrease of tick burdens in the environment can be achieved using conventional and slow release acaricidal formulations applied by spray or powder. As Babesia species are transmitted by
blood products transfusions, it is recommended to screen canine blood donors
for Babesia infection on a regular basis. Non-vectorial transmission of babesiae by dog to dog fighting and blood transfusions is preventable and can be responsible for the spread of babesiosis into previously non-endemic areas. A
vaccine against B. canis is commercially available in some countries in Europe. Vaccines induce partial protection against disease caused by B. canis
manifested by decreased severity of clinical signs, parasitaemia or duration of
clinical disease induced by infection challenge.
FELINE BABESIOSIS
Babesiosis of domestic cats has mostly been reported in South Africa
where infection is mainly due to Babesia felis, a small Babesia sp. that causes anemia and icterus (Ayoob et al., 1010b).
In addition, Babesia cati was reported from a cat in India and sporadic cases of infection in domestic cats by unnamed Babesia parasites have been reported in France, Germany, Thailand and Zimbabwe. Babesia canis infection
in domestic cats with molecular evidence for infection was reported in a study
from Spain and Portugal from three cats. Additionally, infection of domestic
cats with a genetically distinct sub-species of B. canis named B. canis subsp.
presentii was described in Israel.
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REFERENCES
Ayoob, A.L., Hackner, S.G., Prittie, J., 2010a, Clinical management of canine babesiosis. J Vet
Emerg Crit Care (San Antonio) 20, 77-89.
Ayoob, A.L., Prittie, J., Hackner, S.G., 2010b, Feline babesiosis. J Vet Emerg Crit Care (San
Antonio) 20, 90-97.
Birkenheuer, A.J., Neel, J., Ruslander, D., Levy, M.G., Breitschwerdt, E.B., 2004a, Detection
and molecular characterization of a novel large Babesia species in a dog. Vet Parasitol
124, 151-160.
Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2004b, Efficacy of combined atovaquone
and azithromycin for therapy of chronic Babesia gibsoni (Asian genotype) infections in
dogs. J Vet Intern Med 18, 494-498.
Camacho-Garcia, A.T., 2006, Piroplasma infection in dogs in northern Spain. Vet Parasitol 138,
97-102.
Irwin, P.J., 2009, Canine babesiosis: from molecular taxonomy to control. Parasit Vectors 2
Suppl 1, S4.
Irwin, P.J., 2010, Canine babesiosis. Vet Clin North Am Small Anim Pract 40, 1141-1156.
Jacobson, L.S., 2006, The South African form of severe and complicated canine babesiosis: clinical advances 1994-2004. Vet Parasitol 138, 126-139.
Kjemtrup, A.M., Wainwright, K., Miller, M., Penzhorn, B.L., Carreno, R.A., 2006, Babesia
conradae, sp. Nov., a small canine Babesia identified in California. Vet Parasitol 138,
103-111.
Penzhorn, B.L., 2011. Why is Southern African canine babesiosis so virulent? An evolutionary perspective. Parasit Vectors 13, 4:51.
Roncalli Amici, R., 2001, The history of Italian parasitology. Vet Parasitol 98, 3-30.
Sikorski, L.E., Birkenheuer, A.J., Holowaychuk, M.K., McCleary-Wheeler, A.L., Davis, J.M.,
Littman, M.P., 2010, Babesiosis caused by a large Babesia species in 7 immunocompromised dogs. J Vet Intern Med 24, 127-131.
Solano-Gallego, L., Trotta, M., Carli, E., Carcy, B., Caldin, M., Furlanello, T., 2008, Babesia
canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by
means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease. Vet Parasitol 157, 211-221.
Solano-Gallego, L., Baneth, G., 2001. Babesiosis in dogs and cats-Expanding parasitological
and clinical spectra. Vet Parasitol. Epub ahead of print.
Uilenberg, G., 2006, Babesia—a historical overview. Vet Parasitol 138, 3-10.
Yeagley, T.J., Reichard, M.V., Hempstead, J.E., Allen, K.E., Parsons, L.M., White, M.A., Little, S.E., Meinkoth, J.H., 2009, Detection of Babesia gibsoni and the canine small Babesia ‘Spanish isolate’ in blood samples obtained from dogs confiscated from dogfighting
operations. J Am Vet Med Assoc 235, 535-539.
Zahler, M., Rinder, H., Schein, E., Gothe, R., 2000, Detection of a new pathogenic Babesia microti-like species in dogs. Vet Parasitol 89, 241-248.
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Gad Baneth
Jerusalem, Israel
What we really know about
canine and feline hepatozoonosis
Sunday, March 10th 2013 - 9.55 am
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Hepatozoonosis is a vector-borne infection caused by apicomplexan protozoa. In contrast to most tick-borne pathogens that are transmitted via the
tick salivary glands, Hepatozoon species infect vertebartes by ingestion of
arthropod hosts containing infective sporozoites. Two different species of Hepatozoon infect dogs, H. canis globally, and H. americanum in the southern
USA. Clinically, H. canis infection (HCI) varies between being asymptomatic
in dogs with a low parasitaemia, to a severe disease with anemia, profound
lethargy and cachexia in dogs with a large number of circulating parasites. H.
americanum infection (HAI) is manifested mainly by gait abnormalities and
musculoskeletal pain due to myosistis and periosteal bone lesions.
TRANSMISSION
The main vector of H. canis is the brown dog tick Rhipicephalus sanguineus which is found in warm and temperate regions all over the world,
making the potential distribution of H. canis wide. In addition, studies from
Brazil have indicated that H. canis can be transmitted to dogs by the tick Amblyomma ovale. The Gulf Coast tick Amblyomma maculatum is the vector of
H. americanum in North America. A. maculatum’s distribution appears to be
limited to some parts of America therefore possibly restricting the spread of
H. americanum. Both of the Hepatozoon species which infect dogs are transmitted transstadially in their tick vectors. In addition, H. canis has been
shown to be transmitted transplacentally from the dam to its pups, whereas H.
americanum is transitted by predation and ingestion of tissue forms in mammalian host’s tissues
PATHOGENESIS
Hepatozoon sporozoites release from the oocysts after their ingestion in
the dog’s intestine and penetrate the gut wall. The sporozoites invade mononuclear cells and disseminate hematogenously or via the lymph to target organs.
Merogony occurs in the dog’s parenchymal tissues and is followed by gamontogony in leukocytes. The tick, which serves as the definitive host, becomes infected when feeding on a parasitemic dog. Hepatozoon gamonts release from the dog leukocytes within the tick gut and gametogenesis takes
place followed by fertilization and sporogony with the formation of oocysts
in the tick’s hemocoel.
Hepatozoon canis mainly infects the hemolymphoid organs including the
bone marrow, lymph nodes and spleen. H. americanum primarily infects
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skeletal and cardiac muscular tissues and causes myositis and severe lameness. H. canis appears to be well adapted to its canine host, and is often detected in necropsy or on a peripheral blood smear as an incidental finding. H.
americanum induces a violent course of disease in experimentally and naturally occurring infection.
CLINICAL AND LABORATORY FINDINGS
HCI varies from being asymptomatic in apparently healthy dogs to a severe and life-threatening disease in animals with extreme lethargy, cachexia
and anemia. An asymptomatic to mild disease is the most common presentation of the infection and it is usually associated with a low level of H. canis
parasitemia (1-5%), while a severe illness is found in dogs with a high parasitemia often approaching 100% of the peripheral blood neutrophils. High
parasitemia rates are sometimes accompanied by extreme neutrophilia reaching as high as 150,000 leukocytes/ml blood. A seroepidemiological study of
HCI in Israel revealed that 33% of the dogs surveyed had been exposed to the
parasite as indicated by the presence of anti-H.canis antibodies. Only 3% of
the seropositive dogs had detectable blood gamonts and only 1% had severe
clinical signs associated with the infection. A case-control study of dogs admitted to a veterinary hospital in Israel with H. canis parasitemia indicated
that 15% had a high number of circulating parasites (> 800 gamonts/ml) accompanied by elevated body temperature, lethargy, weight loss, anemia and
hyperglobulinemia. Post-mortem findings from some of the dogs with a high
parasitemia revealed hepatitis, pneumonia and glomerulonephritis associated
with H. canis meronts. Meronts of H. canis were also found in the spleen,
bone marrow and lymph nodes. Concurrent HCI and infection with other canine pathogens is common. Co-infections with H. canis reported include: parvovirus, Ehrlichia canis, Toxoplasma gondii and Leishmania infantum. Immune suppression induced by an infectious agent, an immature immune system in young animals or immunodeficient conditions, influence the pathogenesis of new H. canis infections or the reactivation of pre-existing ones.
Treatment with an immunosupressive dose of prednisolone was followed by
the appearance of H. canis parasitemia in dogs with experimental HC.
In contrast to the generally mild disease found in HCI, HAI is almost always a severe disease that leads to debilitation and death. Most dogs diagnosed
with HAI are presented with fever, gait abnormalities, muscular pain induced
by myositis, generalized muscular atrophy and mucopurulent ocular discharge.
The pain can be generalized or localized in the lumbar and cervical spine, or
joints. Gait abnormalities include stiffness, hind limb paresis, ataxia and in36
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ability to rise. A marked neutrophilia is one of the consistent hematologic findings in HAI. Leukocyte counts range from 30,000 to 200,000/ml blood. Serum
biochemical abnormalities include increased alkaline phosphatase activity and
hypoalbuminemia.
DIAGNOSIS
Microscopy: HCI is usually diagnosed by microscopic detection of intracellular H. canis gamonts in stained blood smears. The gamonts are found in
the cytoplasm of neutrophils or monocytes, have an ellipsoidal shape and are
about 11 by 4 micrometers. H. canis meronts found in infected tissues by
histopathology contain elongated micromerozoites arranged in a circle around
a clear central core. This form is often referred to as a “wheel spoke” meront.
H. americanum parasitemia is rare and usually does not exceed 0.1% of
the leukocytes. Confirmation of HAI is commonly carried out by muscle
biopsy and demonstration of parasites in cysts or granulomas. Histopathology of skeletal muscles from dogs with HAI reveals pyogarnulomatous myositis and large round to oval cysts (250-500 micrometer diameter) containing a
central nucleus surrounded by concentric rings of membranes. These cysts are
sometimes referred to as having an “onion peel” appearance due to the structure of the membranes surrounding a core mass. Radiography of the long
bones or pelvis demonstrating periosteal proliferation can be used for screening suspected animals.
Serology: An indirect fluorescent antibody test (IFAT) for anti-H. canis
antibodies was used for epidemiologcial studies in Israel and Japan. An
ELISA for H. canis antibodies has also been developed and used for studies
in Israel and Greece.
PCR: PCR for H. canis in blood has been shown to be a sensitive diagnostic technique. A study from Turkey demonstrated that detection of hepatozoonosis by PCR is by far more sensitive than light microscopy of blood. The
prevalence of hepatozoonosis among 349 dogs was 10.6% by blood smear
evaluation and 25.8% by blood PCR.
FELINE HEPATOZOONOSIS
Hepatozoonosis of domestic cats has been reported from several countries
including: India, South Africa, Nigeria, the USA, Brazil, Israel and France. It
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appears that most feline hepatozoonosis infections are caused by Hepatozoon
felis, However, feline infection can also be caused by H. canis. The vector of
H. felis arecurrently unknown. Hepatozoon felis infection is associated with
infection of muscle tissues.
H. felisn meronts have been identified in the myocardium and skeletal
muscles of cats with hepatozoonosis, and elevated activities of the muscle enzyme CK were found in the majority of cats with hepatozoonosis in a retrospective study of this disease. The level of parasitaemia is usually low in cats
with less than 1% of the neutrophils containing gamonts.
TREATMENT AND PREVENTION
The current treatment protocol for H. canis infection is the administration
of imidocarb dipropionate at 5-6 mg/kg every 14 days until gamonts are no
longer detectable in blood smears. Elimination of H. canis gamonts from the
peripheral blood may require many repeated treatments and PCR studies have
indicated that complete elimination may not be feasible. The prognosis of
treated dogs with a low H. canis parasitemia is generally good even if decrease of parasitemia is slow and requires several repeated imidocarb treatments. The prognosis for dogs with a high parasitemia is good to guarded and
it is sometimes associated with the outcome of a concurrent illness.
Hepatozoon americanum infection is treated with a combination oral therapy of trimethoprim-sulfadiazine (15 mg/kg every 12 hours), pyrimethamine
(0.25 mg/kg every 24 hours), and clindamycin (10 mg/kg every 8 hours) for
14 days. After initial relief from clinical disease signs is obtained, remission
can be prolonged with the oral administration of the coccidiostat decoquinate
at 15 mg/kg mixed in food every 12 hours for two years. Relapse of clinical
disease is common following the discontinuation of treatment.
Prevention of H. canis and H. americanum infection consists of the use of
topical acaricides and environmental parasiticides, prevention of oral ingestion of ticks, and of predation on infected mammal hosts. No commercial vaccines are available for canine hepatozoonosis.
REFERENCES
Baneth G. (2011) Perspectives on canine and feline hepatozoonosis. Veteterinary Parasitology
181, 3-11.
Baneth, G. & Weigler, B. (1997) Retrospective case-control study of hepatozoonosis in dogs in
Israel. Jouranl of Veterinary Internal Medicine 11, 365-370.
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Baneth. G., Samish, M., Aroch, I., Alekseev, Y. & Shkap, V. (2001) Transmission of Hepatozoon canis to dogs by naturally fed or percutaneously injected Rhipicephalus sanguineus
ticks. Journal of Parasitology 87, 606-611.
Baneth, G., Mathew, J.S., Shkap, V., Macintire, D.K., Barta, J.R., Ewing, S.A. (2003) Canine
hepatozoonosis: two disease syndromes caused by separate Hepatozoon species. Trends
in Parasitology 19, 27-31.
Baneth, G., Samish, M., Shkap, V. (2007). The life cycle of Hepatozoon canis (Apicomplexa:
Adeleorina) in the tick Rhipicephalus sanguineus and the Domestic dog (Canis familiaris). Journal of Parasitology. 93, 283-299.
Ewing, S. A., DuBois, J. G., Mathew, J.S. & Panciera, R.J. (2002) Larval Gulf Coast ticks (Amblyomma maculatum) [Acari: Ixodidae] as host for Hepatozoon americanum [Apicomplexa: Adeleorina]. Veterinary Parasitology 103, 43-51.
Ewing, S.A., Panciera, R.J. (2003) American canine hepatozoonosis. Clinical Microbiology
Reviews 16, 688-697.
Forlano, M., Scofield, A., Elisei, C., Fernandes K.S., Ewing, S.A., Massard, C.L. (2005). Diagnosis of Hepatozoon spp. in Amblyomma ovlae and its experimental transmission in domestic dogs in Brazil. Veterinary Parasitology 134, 1-7.
Gavazza, A., Bizzeti, M., Papini, R. Observations on dogs found naturally infected with Hepatozoon canis in Italy. (2003). Reveue Medicine Veteterinaire 159, 565-571.
Karagenc, T.I., Pasa, S., Kirli, G., Hosgor, M., Bilgic, H.B., Ozon, Y.H., Atasoy, A., Eeren, H.
(2006). A parasitological, Molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey. Veterinary Parasitology 135, 113-119.
Macintire, D.K., Vincent-Johnson, N.A., Kane, C.W., Lindsay, D.S., Blagburn, B.L. & Dillon,
A.R. (2001) Treatment of dogs infected with Hepatozoon americanum: 53 cases (19891998). Journal of American Veterinary Medical Association 218, 77-82.
Vincent-Johnson, N. A., Macintire, D.K., Lindsay, D.S., Lenz, S.D., Baneth, G., Shkap, V. &
Blagburn, B.L. (1997) A new Hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in North America. Journal of Parasitology 83, 11651172.
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Giovanni Barsotti
Med Vet, Dr Ric, SCMPA, Pisa, Italy
Lesioni oculari associate
alla Leishmaniosi canina
Venerdì, 8 Marzo 2013 - ore 11.55
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La letteratura veterinaria riporta una prevalenza di affezioni oftalmiche in
cani con leishmaniosi sistemica compresa tra il 16 e l’80% (Roze, 1986; Slappendel, 1988; Molleda et al., 1993; Ciaramella et al., 1997; Koutinas et al.,
1999; Peña et al., 2000). Questo ampio divario può derivare dal fatto che la
maggior parte degli studi fino ad oggi pubblicati sono di tipo retrospettivo, con
semplice valutazione delle cartelle cliniche degli animali malati e quindi alcuni dati possono essere assenti o erroneamente interpretati. Inoltre alcune pubblicazioni, seppur importanti da un punto di vista scientifico, riportano la prevalenza di tutte le manifestazioni cliniche della leishmaniosi, comprese quelle
oftalmiche, senza però specificare come siano stati ottenuti i risultati. Ne consegue che, se non è stata effettuata una visita oculistica specialistica completa,
alcune patologie subcliniche o con segni clinici non evidenti (ad esempio:
uveite posteriore, emorragie retiniche) possano non essere state diagnosticate.
Le affezioni oftalmiche osservate nella Leishmaniosi canina sono da ascrivere, come per altri tessuti, a un danno diretto da parte del parassita e/o a danno conseguente al deposito di immunocomplessi (Garcia-Alonso et al., 1996;
Peña et al., 2008).
Secondo alcuni autori i settori oculari maggiormente coinvolti in cani con
leishmaniosi sistemica sarebbero gli annessi e la superficie oculare (blefarite,
congiuntivite, cheratocongiuntivite) (Roze, 1986; Slappendel, 1988; Molleda
et al., 1993; Ciaramella et al., 1997) mentre secondo altri la flogosi cronica
dell’uvea anteriore rappresenterebbe il reperto oftalmico più frequente (Peña
et al., 2000).
In particolare Molleda et al. (1993) riportano che l’80.49% di 41 cani con
leishmaniosi presentava lesioni oftalmiche e di queste circa il 75% coinvolgeva la congiuntiva. Le affezioni congiuntivali diagnosticate sono state in ordine
di frequenza: congiuntivite, cheratocongiuntivite secca, blefarocongiuntivite e
cheratocongiuntivite mucopurulenta. Gli altri distretti oculari coinvolti erano la
cornea, le palpebre, il fondo oculare e l’uvea anteriore. L’esame istopatologico degli occhi di 23/41 soggetti valutati ha messo in evidenza nel 96.65% dei
casi un infiltrato linfoplasmacellulare da lieve ad abbondante a carico di diverse strutture oculari tra cui la terza palpebra, la cornea, il limbo corneosclerale
e l’uvea anteriore e in pochi casi la presenza del parassita. Nello studio manca
però la correlazione tra i reperti istopatologici riscontrati e la tipologia dei segni clinici oftalmici e gli autori si limitano ad affermare che il numero di animali che manifestano un’evidenza istopatologica di malattia parassitaria è superiore a quello dei soggetti con affezioni cliniche evidenti, postulando che una
parte dei segni clinici più lievi possa essere passata inosservata.
Peña et al. (2000) in uno studio clinico riportano, invece, che circa il 25%
di 105 cani con leishmaniosi sistemica presentava oculopatie. Nella maggior
parte dei casi il coinvolgimento era bilaterale e molti cani manifestavano pa41
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tologie in più di un settore oculare. Le affezioni oculari maggiormente riscontrate erano in ordine di frequenza: uveite anteriore, congiuntivite, cheratocongiuntivite, alopecia perioculare e blefarite diffusa. L’uveite anteriore
rappresenta sicuramente la patologia più frequente, se però, valutando criticamente i risultati dello studio, sommiamo le singole lesioni oftalmiche che
coinvolgono gli annessi oculari e la cornea, ci rendiamo conto che, anche in
questo caso, le patologie degli annessi e della superficie oculare sono superiori a quelle intraoculari.
Recentemente studi istopatologici effettuati su 120 occhi di 60 cani affetti
da leishmaniosi e sottoposti a eutanasia hanno evidenziato che la congiuntiva
è il tessuto dove più frequentemente è possibile reperire il parassita con tecniche immunoistochimiche. Inoltre sempre la congiuntiva è risultata il tessuto oftalmico maggiormente colpito da flogosi, quest’ultima caratterizzata prevalentemente da un quadro istologico di infiltrazione perivascolare linfoplasmacellulare e in misura minore di infiltrazione perivascolare linfoplasmacellulare con presenza di macrofagi, infiltrazione diffusa linfoplasmacellulare
con macrofagi e infine di tipo piogranulomatoso (Peña et al., 2008).
Se riassumiamo quindi l’esperienza clinica dei vari autori che hanno descritto i segni clinici oculari della Leishmaniosi canina (Roze, 1986; Slappendel, 1988; Molleda et al., 1993; Ciaramella et al., 1997; Koutinas et al., 1999;
Peña et al., 2000) è possibile affermare che le patologie degli annessi e della
superficie oculare e l’uveite anteriore rappresentano le manifestazioni più frequenti. Tra le patologie degli annessi e della superficie oculare repertiamo: blefarite (desquamativa, nodulare, ulcerativa), congiuntivite di vario tipo, cheratocongiuntivite e cherato-congiuntivite secca. Quest’ultima sembra dovuta alla
ritenzione del secreto delle ghiandole lacrimali secondaria all’infiltrazione infiammatoria intorno ai dotti escretori che ne causa ostruzione progressiva (Naranjo et al., 2005). L’uveite anteriore può essere diffusa o nodulare e determinare complicazioni quali cataratta e glaucoma. Altri segni clinici oftalmici in
corso di Leishmaniosi canina sono l’uveite posteriore, l’episclerite sia nodulare sia diffusa, la sclerite e più raramente la cellulite orbitale. In corso di Leishmaniosi canina è possibile osservare a livello oculare alterazioni riconducibili a ipertensione sistemica quali: emorragia retinica e distacco retinico parziale o completo (Cortadellas et al., 2006; Peña et al., 2008). Si possono riscontrate, inoltre, emorragia intra-iridea, ifema e tortuosità vascolare retinica.
È importante sottolineare che spesso più segni clinici di leishmaniosi oculare coesistono e che nessuna manifestazione oculare è da considerarsi patognomonica di leishmaniosi ma deve rientrare in un attento piano diagnostico differenziale. La prognosi quoad functionem dell’apparato visivo è sempre riservata
anche in presenza di segni clinici lievi o moderati, soprattutto nelle affezioni intraoculari. Talvolta le condizioni cliniche locali si possono aggravare in corso di
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terapia eziologica a causa della morte dei parassiti che può causare una grave
uveite o aggravarne una già presente (Peña et al., 2000). La terapia prevede
l’impiego di farmaci sistemici anti-Leishmania in associazione alla terapia sintomatica delle diverse alterazioni oculari diagnosticate, considerando che è relativamente frequente osservare a livello oculare patologie croniche o che presentano periodiche riacutizzazioni. L’impiego dei corticosteroidi sistemici e locali, questi ultimi in assenza di lesioni ulcerative corneali, è essenziale nelle forme flogistiche intraoculari al fine di evitare lo sviluppo di complicanze quali il
glaucoma che può determinare la perdita della funzione visiva.
BIBLIOGRAFIA ESSENZIALE
Ciaramella P., Oliva G., De Luna R., Gradoni L., Ambrosio R., Cortese L., Scalone A., Persechino A. “A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum”. Veterinary Record 141: 539-543, 1997.
Cortadellas O., del Palacio M.J., Bayón A., Albert A., Talavera J. “Systemic hypertension in
dogs with leishmaniasis: prevalence and clinical consequences”. Journal of Veterinary Internal Medicine 20: 941-947, 2006.
Koutinas A.F., Polizopoulou Z.S., Saridomichelakis M. N., Argyriadis D., Fytianou A., Plevraki K.G.: “Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989-1996)”. Journal of the American Animal Hospital Association 35: 376-383, 1996.
Garcia-Alonso M., Blanco A., Reina D,Serrano F.J., Alonso C. “Immunopathology of the uveitis in canine leishmaniasis”. Parasite Immunology 18: 617-623, 1996.
Molleda J.M., Novales M., Ginel P.J., Fernandez A., Lopez R.: “Clinical and histopatological
study of eye in canine leishmaniosis”. Israel Journal of Veterinary Medicine 48: 173-178,
1993.
Naranjo C., Fondevila D., Leiva M., Roura X., Peña T.: “Characterization of lacrimal gland lesions and possible pathogenic mechanisms of keratoconjunctivitis sicca in dogs with leishmaniosis”. Veterinary Parasitology 133: 37-47, 2005.
Peña M.T., Roura X., Davidson M.G.: “Ocular and periocular manifestations of leishmaniasis
in dogs: 105 cases (1993-1998)”. Veterinary Ophthalmology. 3: 35-41, 2000.
Peña M.T, Naranjo C., Klauss G., Fondevilla D., Leiva M., Roura X., Davidson M.G., Dubielzing R.R.: “Histopatological Features of Ocular Leishmaniosis in the dog”. Journal of
Comparative Pathology 138: 32-39, 2008.
Roze M.: “Manifestacions Oculaires de la Leishmaniose Canine”. Recueil de Medicine Vaterinaire 19-26, 1986.
Slappendel R.J.: “Canine leishmaniasis. A review based on 95 cases in The Netherlands”. Veterinary Quarterly 10: 1-16, 1988.
Indirizzo per la corrsipondenza:
Dipartimento di Scienze Veterinarie - Università di Pisa
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Erika Carli
Med Vet, Padova, Italy
Che cosa sappiamo
del Cytauxzoon in Europa?
Domenica, 10 Marzo 2013 - ore 09.00
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La cytauxzoonosi è un’infezione protozoaria trasmessa da zecche che colpisce i felini domestici e selvatici. L’agente responsabile della malattia è Cytauxzoon felis (C. felis) e, nel gatto, è stato descritto per la prima volta in Missouri nel 1976. Il Cytauxzoon fa parte dell’ordine Piroplasmidae e della famiglia Theileriidae.
La maggior parte delle segnalazioni è stata fatta negli Stati Uniti (stati del
centro, centro sud e sud est). In Europa, le infezioni da C. felis sono state riportate molto raramente. In Germania è stato descritto il caso di una tigre, che
era nata e vissuta in uno zoo, e che ha sviluppato un’infezione fatale un anno
dopo l’introduzione nel suo ambiente di alcune linci provenienti dagli Stati
Uniti. In Turchia è stata osservata in un gatto di Van: in questo caso la diagnosi si basava sulla sola valutazione morfologica di uno striscio di sangue
senza conferma mediante indagine molecolare.
La lince americana è considerata il reservoir naturale dell’infezione per i
felini selvatici e domestici. Oltre alla lince, il ghepardo, il puma, il leone e la
tigre bianca hanno presentato l’infezione e potrebbero essere dei potenziali
reservoir. I vettori imputati della trasmissione sono le zecche, in particolar
modo Amblyomma americanum e Dermacentor variabilis che sperimentalmente hanno trasmesso l’infezione ai gatti. Il ciclo di C. felis prevede una fase tissutale, con localizzazione del parassita a livello dei fagociti mononucleati associati all’endotelio dei vasi di vari organi (schizonti), e una fase eritrocitaria (merozoiti). Durante la fase tissutale, la rapida moltiplicazione del
parassita si associa alla comparsa dei gravi segni clinici che caratterizzano
questa malattia nel gatto. I segni clinici sembrano essere la conseguenza dell’ostruzione meccanica del flusso ematico (soprattutto attraverso i polmoni) e
della produzione di sostanze tossiche, vasoattive e pirogene da parte del parassita. L’emolisi e la rimozione dei globuli rossi parassitati, con conseguente comparsa di anemia, sono invece secondari alla localizzazione intraeritrocitaria del patogeno. La coagulazione intravasale disseminata spesso complica il quadro clinico.
Nel gatto, la cytauxzoonosi si manifesta come una malattia grave, caratterizzata da evoluzione rapida, spesso con esito fatale. Il periodo di prepatenza
è di 2-3 settimane. I segni clinici riportati sono generalmente aspecifici e
comprendono, tra gli altri, depressione, letargia, dispnea, disidratazione,
anoressia, vomito, ittero, splenomegalia, epatomegalia e febbre. Per quanto
riguarda le alterazioni di laboratorio, frequentemente si osserva anemia normocitica normocromica non rigenerativa associata ad un conteggio leucocitario variabile. Nella fase finale della malattia si possono riscontrare gravi leucopenie e trombocitopenie. Spesso sono presenti importati incrementi della
bilirubina sierica e urinaria. Nella maggior parte dei soggetti colpiti, la morte
sopraggiunge rapidamente (anche in meno di 5 giorni dalla comparsa dei sin45
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tomi). Recentemente però, negli Stati Uniti sono stati documentati sporadici
casi di sopravvivenza dopo l’infezione. Inoltre sono stati segnalati soggetti
asintomatici con infezioni persistenti. Nei felini selvatici l’infezione generalmente è caratterizzata da eritroparassitemia subclinica e i casi di malattia conclamata e fatale sono molto rari.
L’infezione da C. felis si sospetta sulla base del reperimento del parassita in sede intraeritrocitaria, mediante valutazione morfologica di uno striscio di sangue, o negli schizonti in reperti citologici o istologici provenienti da vari tessuti (fegato, polmoni, milza, utero, midollo osseo, linfonodi). A
livello tissutale si osserva l’accumulo di fagociti mononucleati contenenti
schizonti in diverse fasi di sviluppo soprattutto in corrispondenza del lume
di vasi venosi che possono andare incontro a parziale o completa ostruzione. Nei globuli rossi, il C. felis appare come un organismo di forma da ovale a rotondeggiante, con diametro di circa 0.5-0.8 m, con nucleo eccentrico
intensamente basofilo e citoplasma lievemente basofilo. Gli inclusi più
spesso sono singoli, talvolta sono appaiati. L’entità della parassitemia varia
da soggetto a soggetto e in base alla fase della malattia e tende ad aumentare con il progredire del quadro clinico. Mediante l’osservazione microscopica non è possibile distinguere C. felis da altri piccoli piroplasmi quali Babesia sp. e Theileria sp. Occorrono ulteriori indagini di biologia molecolare (PCR e sequenziamento) per individuare chiaramente il patogeno coinvolto ed emettere la diagnosi definitiva.
Ancora non è disponibile una terapia sicuramente efficace per il trattamento della cytauxzoonosi. È stato descritto l’uso di vari principi attivi come
ad esempio atovaquone, imidocarb, diminazene diaceturato, azitromicina, enrofloxacina da soli o in associazione. Spesso, in base alla sintomatologia e alle complicazioni osservate nei soggetti malati, alla terapia antiparassitaria si
associa la somministrazione di eparina, trasfusioni o altri farmaci.
Recentemente sono state descritte altre specie di Cytauxzoon che, dal punto di vista molecolare, sono simili a C. felis, ma non completamente e che
sembrerebbero costituire un gruppo filogenetico a sé stante. Nel 2003 è stata
segnalata per la prima volta in alcuni gatti di Pallas, importati in Oklahoma
dalla Mongolia, un’infezione da parte di un Cytauxzoon che è stato denominato Cytauxzoon manul (C. manul). In seguito in Spagna, sono state descritte
infezioni da Cytauxzoon sp. simili a C. manul nelle linci iberiche e in un gatto. In Francia è stato segnalato un gatto che presentava una coinfezione da Hepatozoon canis e Cytauxzoon sp. Per quanto riguarda l’Italia, la prima segnalazione è del 2011 e riguarda la descrizione di un focolaio d’infezione da Cytauxzoon sp. a Trieste. Questo lavoro è iniziato con l’osservazione di inclusi
eritrocitari ascrivibili a piroplasmi in 3 gatti. L’indagine molecolare, eseguita
successivamente mediante PCR e sequenziamento di un frammento di 412 pa46
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ia di basi del gene 18S rRNA, ha portato alla diagnosi di infezione da Cytauxzoon sp. in tutti i soggetti. I gatti studiati avevano segni clinici differenti
(problemi dermatologici, ematologici e neurologici) ma, in comune, avevano
la provenienza (Trieste). In seguito sono stati raccolti campioni di sangue da
gatti di proprietà e da gatti di colonia che risiedevano nella città ed è stata fatta una ricerca per Cytauxzoon sp. mediante valutazione morfologica di uno
striscio di sangue, PCR e sequenziamento. Sono stati individuati 27/118 gatti
positivi all’infezione con una prevalenza del 23%. Le indagini molecolari e filogenetiche eseguite sui campioni positivi hanno evidenziato un’omologia del
99% con le sequenze depositate in GenBank per i Cytauxzoon segnalati in
Mongolia, Spagna e Francia e del 93% con quelle di C. felis. Sono state quindi analizzate informazioni riguardanti segnalamento, stile di vita, segni clinici e di laboratorio raccolti dai gatti coinvolti nello studio. Si è osservato che
mentre nei casi d’infezione da C. felis nel gatto, l’eritroparassitemia spesso si
associava a segni clinici, nei gatti infetti da Cytauxzoon sp. più spesso era presente in soggetti apparentemente sani. I gatti di colonia in virtù del loro stile
di vita (più esposti a vettori e reservoir, nessuna profilassi per gli ectoparassiti, stati nutrizionale e clinico più scadenti) risultavano più suscettibili all’infezione rispetto ai gatti di proprietà. Inoltre, non è stata rilevata associazione
statisticamente significativa tra infezione da Cytauxzoon sp. e razza, sesso,
età, presenza di vettori (zecche e/o pulci), stato clinico, alterazioni di laboratorio, positività per FIV e/o FeLV e tasso di mortalità. Particolarmente interessante è stata la mancata associazione dell’infezione con la presenza di anemia (segno clinico ben descritto in corso d’infezione da C. felis) e la mortalità (particolarmente elevata nei soggetti colpiti da cytauxzoonosi da C. felis).
Dai dati raccolti l’infezione da Cytauxzoon sp. sembra essere principalmente
di tipo subclinico.
Non è da escludere che in corso di malattie concomitanti e/o stati immunosoppressivi l’infezione cronica possa diventare manifesta, evolvendo in cytauxzoonosi conclamata. Per quanto riguarda i vettori e i reservoir di Cytauxzoon sp. non sono disponibili informazioni, ma probabilmente la lince e le
zecche che si trovano sul territorio italiano sono coinvolte nel ciclo del parassita. Oltre al focolaio descritto a Trieste, si segnalano altri casi d’infezione
diagnosticati in zone del nordest e centro Italia (dati non pubblicati). Inoltre i
primi risultati di uno studio epidemiologico ancora in corso, indicherebbero
una prevalenza per l’infezione da Cytauxzoon sp. in Italia intorno all’1.96%.
I dati descritti rappresentano le prime osservazioni riguardanti un parassita del
quale si conosce poco ma che si può incontrare nella pratica clinica. Cytauxzoon sp. è, perciò, un patogeno emergente da includere nelle diagnosi differenziali di malattia, soprattutto nei soggetti che vivono nelle zone in cui sono
presenti i vettori dell’infezione.
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Filipe Dantas-Torres
DVM, MSc, DSc, PhD, FRES, Dipl EVPC, Brazil
Changing epidemiology
of vector-borne diseases
Saturday, March 9th 2013 - 12.10 am
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Vector-borne diseases are diseases caused by pathogens that are transmitted by arthropod vectors, such as mosquitoes, ticks, phlebotomine sand flies,
biting midges, black flies, fleas, lice, and kissing bugs. Mosquitoes and ticks
are the most important arthropod vectors of pathogens to humans and domestic animals. For instance, mosquitoes are responsible for the transmission of
viruses, protozoa, and filarioids. Ticks may also transmit a wide range of
pathogens, including bacteria, viruses, and protozoa. Phlebotomine sand flies
are the main vectors of Leishmania parasites, but may also transmit viruses
and bacteria.
The incidence of several vector-borne diseases (e.g., leishmaniasis, dirofilariosis, and thelaziosis) is on the rise. The increased incidence and expanded
distribution of vectorborne disease has been attributed to a range of abiotic
and biotic factors, including climate changes, increased susceptibility of
hosts, increased population movements, changes in human behavior, and relaxation of control measures due to economic crisis.
For instance, the epidemiology of vector-borne diseases is greatly influenced by vector population ecology, which in turn is driven by factors such as
climate variables, vegetation coverage, and host population density. Changes
in incidence and distribution may also be a result of increased human population movements and animal trafficking.
Environmental changes (e.g., deforestation) and major catastrophic events
(e.g., earth quakes, tsunami) may also impact on the epidemiology of vectorborne diseases. Moreover, the increasing proportions of young people in
countries of emerging economies as opposed to an increasing proportion of
older people in the developed economies may also increase the susceptibility
of human populations to pathogens such as Leishmania infantum.
VECTOR-BORNE PROTOZOA
Changes in the epidemiology of several vector-borne protozoa have been
documented in recent years. For instance, human malaria has traditionally
been associated to four human-adapted Plasmodium species: P. vivax, P. falciparum, P. malariae, and P. ovale. However, a fifth species namely P.
knowlesi has been implicated in human disease in southeastern Asia, particularly in Malaysian Borneo, Thailand, Myanmar, and the Philippines. Plasmodium knowlesi is a simian parasite, whose principal natural hosts are pigtailed and the long-tailed macaques. The infection is transmitted by different
anopheline species belonging to the Anopheles leucosphyrus group. The infection by P. knowlesi is common in people that have spent some time in jungles in southeastern Asia. While currently humans are considered to be inad49
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vertent hosts of P. knowlesi, the risk of host switch events (i.e., changes from
macaque-to-human to human-to-human transmission) is real and should be
taken into account.
Chagas disease is caused by Trypanosoma cruzi, which is primarily transmitted by species of kissing bugs. Public control campaigns have greatly reduced the vectorial transmission of T. cruzi in Latin America. However, recent
studies have demonstrated the existence of domestic populations of triatomine
vectors in southeastern Brazil, which demonstrates the risk of vectorial transmission in this region. The existence of wild populations of T. infestans infected with T. cruzi in Chile also indicates the risk of re-introduction of this
vector into the domestic environment in certain areas. Changes in land use,
deforestation, and human population movements may also increase the contact between humans and potential wild triatomine vectors. Non-vectorial
transmission of T. cruzi via blood transfusion, congenital and organ transplantation has also been documented. Remarkably, oral transmission of T.
cruzi has become a common phenomenon and outbreaks of Chagas disease
transmitted by accidental ingestion of contaminated sugar cane, guava or açaí
juices have been reported in South America.
Indeed, most new cases of Chagas disease in Brazil have been associated
to oral transmission.
Babesiosis is a disease caused by species of Babesia, which are primarily
transmitted by hard ticks. These parasites may infect and cause life-threatening diseases in domesticated animals and humans. Cases of human babesiosis
in North America have been associated with Babesia microti whereas cases in
Europe have been attributed to Babesia divergens. However, several Babesia
species have recently been implicated in human disease. In particular,
Babesia duncani and B. divergens-like organisms have been recognized as
pathogens of medical significance in the United States. Moreover, autochthonous cases of B. microti infections have been diagnosed in Taiwan, Japan and
Europe. Babesia venatorum (formerly called EU1), which is probably a parasite of deer, was involved in the first documented cases of human babesiosis
in Italy, Austria, and Germany. The changing epidemiology of human
babesiosis is of concern, also considering the severity of the disease in humans, with case fatality rates reaching 45% in B. divergens infections.
Leishmaniosis is a variegate group of diseases caused species of Leishmania, which are through the bite of phlebotomine sand flies. The ecology and
distribution of phlebotomine sand flies are strongly dependent on climate and
other environmental variables. Indeed, climate changes may potentially extend the period of activity of phlebotomine sand flies and also allow their establishment in new areas where they are currently absent. Recent studies have
reported the introduction of leishmaniasis into previously free areas. For in50
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stance, cases of canine leishmaniasis in Italy have been traditionally reported
in southern regions, but several new foci have recently been detected in northern Italy. Similarly, canine leishmaniasis is now well established in South
America as far south as northern Argentina. The expanding distribution of canine leishmaniasis may be associated to changes in vector distribution due to
the extension of suitable areas as well as to introduction of infected dogs into
free areas.
VECTOR-BORNE HELMINTHES
There has been a clear increase in cases of human and canine dirofilariosis by D. immitis in Europe, which might be related to changes in vector and
dog population dynamics. For example, a southward spread of canine dirofilariosis has been documented in Italy and changes in distribution have been
attributed to the introduction of competent vectors into new areas. Indeed, the
spread of Aedes albopictus throughout Italy has contributed to this process.
This mosquito is a competent vector of both D. immitis and D. repens and is
well adapted to climatic conditions in Mediterranean countries. Thus, it may
be speculated that new foci of canine and human dirofilariosis are likely to be
detected in Europe.
Another example of emerging vector-borne pathogen is represented by
Thelazia callipaeda, the oriental eyeworm. Following these first reports in
Italy, new cases of thelaziosis have also been recorded in France and Germany
in dogs that had spent some time, mostly during summer, in northern Italy.
Recently, autochthonous cases of thelaziosis affecting dogs and cats have
been described in southwestern France and Switzerland. Predictive models indicate the existence of suitable areas for the establishment of the vector Phortica variegata in many European countries where T. callipaeda is currently
considered to be absent.
VECTOR-BORNE BACTERIAL DISEASES
Tick-borne bacteria are among the most important emerging pathogens of
humans in present days. Cases of Lyme disease and Rocky Mountain spotted
fever are on the rise in the United States. Moreover, a case of human rickettsiosis by Rickettsia massiliae, originally described in France and thought to
be restricted to Europe, has been detected in Argentina. This exemplifies that
such microorganism recognize no frontiers and may be introduced into new
areas if animals and vectors such as ticks are not properly controlled. Cases
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of human ehrlichiosis caused by Ehrlichia canis have also been detected in
South America and indicates the potential zoonotic role of this widespread canine pathogen.
FINAL THOUGHTS
The changing scenario of vector-borne diseases will challenge physicians
and veterinarians worldwide and require changes in terms of management and
control practices. Indeed, medical physicians and veterinary practitioners
need to be aware of these changes and of new practices in terms of diagnosis,
treatment and control of vector-borne diseases. Indeed, awareness is the first
step toward early diagnosis, better management and more efficient prevention
of vector-borne diseases. As a consequence, the lack of awareness may represent a major constraint on their successful management and control of vectorborne diseases in previously non-endemic areas. Permanent monitoring and
research and development of improved tools for the detection, treatment and
control of vector-borne diseases should be a priority.
Address for correspondence:
Department of Veterinary Medicine, University of Bari, Str Prov per Casamassima km 3,
70010 Valenzano/BA, Italy; 2Department of Immunology, Aggeu Magalhães
Research Institute, Av Prof Moraes Rego s/n, 50670420 Recife/PE, Brazil
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Laura Ordeix
Esteve, Lda Vet, Dipl ECVD,
Barcelona, Spain
Laia Solano-Gallego
Lda Vet, PhD, Dipl ECVCP,
Barcelona, Spain
What is new on host-parasite
interaction and immunity
in canine Leishmaniosis?
Friday, March 8th 2013 - 09.40 am
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INTRODUCTION
Infectious diseases rarely result from the deliberate activities of a malicious microorganism. In most cases, disease occurs because of the host’s reaction to the infection. Well-adapted parasites, for instance, have evolved in
such way that their presence in the host is scarcely noticed. Indeed, in many
cases, the presence of parasites come to our attention only when they are present in unusually large numbers or when they damage critical organs by accident. A successful parasite evades host’s immune response to permit parasite
survival, while at the same time allowing other responses to proceed and thus
preventing the death of the host from other infections.1
The importance of immunity in determining the course and outcome of a
protozoan disease is best seen in canine leishmaniosis due to Leishmania infantum.1 Not every dog naturally or experimentally infected with Leishmania
develops disease. In fact, the seroprevalence reported in dogs in the Mediterranean basin ranges from 5 to 30% depending on the region.
Surveys employing other detection methods, such as amplification of
Leishmania DNA from different tissues or by detection of specific anti-Leishmania cellular immunity, have revealed even higher infection rates approaching 70% in some foci. 2,3 Therefore, most dogs in these areas appear to have
chronic infection that may be life-long, but only a low proportion of dogs develop severe disease.4
The fate of Leishmania infection in dogs is linked to the host immune responses and to the persistence and multiplication of the parasite. A broad
range of clinical manifestations and immune responses have been described
for canine leishmaniosis. Canine L. infantum infection can manifest as a
chronic subclinical infection, self-limiting disease, or non-self limiting illness.5, 6 In addition, several degrees of disease severity are found in dogs ranging from mild to severe fatal disease with different clinical outcomes, prognosis and treatment options. The two extremes of this clinical spectrum are
represented by “resistant” dogs and “susceptible” dogs.4
Clinically “resistant” dogs
General speaking, the infected dogs either remain healthy or develop a
mild, self-limited cutaneous disease.
These resistant dogs display a weak antibody response but strong and effective Th1-like response. This protective CD4+ T-cell-mediated immune response is characterized by production of Th1 cytokines such as IFN-g, IL-2
and TNF-a, which induce anti-Leishmania activity by apoptosis of parasites
in macrophages via NO metabolism and, thus capable of controlling infec54
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tion. They have low or negative antibody levels, mount a strong delayed hypersensitivity response and low parasite loads.
This subclinical infection is not necessarily permanent, and factors such as
immunosuppressive conditions or concomitant disease can break the equilibrium and lead to the progression of clinical disease in dogs.4
Clinically “susceptible” dogs
They mount a Th2-like response characterized by a marked humoral immune response and high antibody levels but reduced cell-mediated immunity
and high parasites loads. Parasites continue to multiply into macrophages.
These macrophages spread throughout the body from the skin to the local
lymph node, spleen, and bone marrow within a few hours, resulting in a disseminated infection and generalized disease characterized initially by lymphadenomegaly, splenomegaly and hyperglobulinemia. They show polyclonal B cell activation and they develop lesions associated with type II and type
III hypersensitivity, such as immune-mediate hemolytic anemia, thrombocytopenia, glomerulonephritis, uveitis, vasculitis, polyarthritis and synovitis.
Serum anti-histone antibodies have been associated with glomerulonephritis
in dogs with leishmaniosis.1,4
It is important to highlight that although the adaptive immune response in
dogs with a Th1 or Th2 responses has been investigated in several studies4, 6,
the mechanisms that underlie and determine these polarized responses are still
poorly understood. The innate immune response has a relevant role in protection against the parasite besides switching on the adaptive response and the
components of innate and adaptive immunity engage in a range of interactions
that is remarkably diverse and complex.7
We focus here on host-parasite interactions and innate immune responses
that participate in the pathogenesis of leishmaniosis.
NEUTROPHILS
When Leishmania promastigote are inoculated by a sand fly into the skin
of dogs, they are rapidly phagocytized by neutrophils. Neutrophils have
emerged as an important cell during Leishmania infection. Recent studies
have confirmed neutrophils as the first cells recruited to and infected in the
dermal site of inoculation. However, the role of neutrophils in either promoting or suppressing host immunity remains controversial.8
Neutrophil infiltration at the site of Leishmania inoculation appears to be
a multifactorial event. The wound repair and sterilization response triggered
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as a result of the tissue damage caused by the sand fly bite is sufficient to drive
the early recruitment of neutrophils. Moreover, it has been demonstrated that
Leishmania promastigotes produce a neutrophil chemotactic factor (LCF) and
induce IL-8 secretion by human neutrophils, a process that might amplify
neutrophil recruitment.8
The fate of ingested parasites is a key issue that remains to be clearly resolved. There is evidence for early killing by neutrophils, attributable to the
oxygen metabolites generated by the phagocytosis-induced respiratory burst.
By contrast, there is conclusive evidence for the survival of L. major promastigotes following their rapid uptake by neutrophils in the skin. Intracellular survival following capture by neutrophils would be consistent with the expression of promastigote-derived molecules, including the cell surface
lipophosphoglycan (LPG) and tartrate-resistant acid phosphatase, that have
been shown to inhibit lysosome fusion and/or the respiratory burst and super
oxide anion production in mouse or human neutrophils.8 Some studies concluded that most of the infected, apoptotic neutrophils lytically released viable parasites may be better adapted for intracellular survival and growth following their uptake by macrophages. The engulfment of infected and uninfected cells or apoptotic bodies by macrophages may inhibit the activation of
infected macrophages in the inflammatory site (the “Trojan horse” model).
The initial infection of dermal DCs appears to take place primarily in the context of their capture of parasitized neutrophils. This encounter strongly reduces their priming capacity and consequently delays the immune response
against the parasite until the acute neutrophilic response is resolved. 9 Thus in
the most relevant inflammatory setting initiated by the bite of an infected sand
fly, in which the acute neutrophilic response is especially massive, localized
and sustained, neutrophils can be shown to suppress immunity and promote
the early infectious process.9
NATURAL KILLER CELLS
Another cell type that represents one of the first lines of defence in the
immune response after invasion of Leishmania parasites is natural killer cells
(NKcells). These cells can produce IFN- that will activate macrophages to
eliminate Leishmania. Absence or low number of NKcells, and impairment
of NK cells response were documented in lesions from human patients with
cutaneous leishmaniosis. The possible explanation for this result, can be the
fact that direct contact points found between Leishmania promastigotes and
naïve human NKcells causes immediate destruction of NKcells in a nonapoptotic way. Indirect evidence of the role of NKcells in the protective im56
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mune response against Leishmania comes from the observation that, in humans with subclinical infection as well as humans without exposure to
Leishmania, NKcells are the main source of IFN- upon stimulation in vitro
with L. aethiopica.10
MACROPHAGES AND DENDRITIC CELLS
The promastigotes transform as amastigotes within phagolysosomes,
which separate them from the host cell defence mechanisms, of infected
macrophages and dendritic cells (after direct phagocytosis or clearance of infected apoptotic neutrophils). Classically activated macrophages secrete IL12, produce oxygen reactive species and are the main cells involved in intracelular parasite killing.
Nitric oxide (NO) produced by canine macrophages has been found to be
the principal effector molecule mediating intracellular killing of Leishmania
amastigotes by apoptotic cell death controlled by proteasome inhibitors.4
However, amastigotes may scape to intracellular destruction as a result of
multiple mechanisms. For instance, Leishmania lipophosphoglycans delays
phagosome maturation, preventing the production of NO and inhibiting
macrophages responses to cytokines. Moreover, the parasite also reduces the
antigen-presenting ability of dendritic cells (essential for an effective immune
response against most pathogens) by suppressing MHC II expression.1
The innate, or nonspecific, immune response, as stated above, is the first
line of defence encountered by Leishmania parasites when entering the susceptible host. In this context, the innate immune system senses the presence
of invading microbes through specific receptors such as Toll-like receptors
(TLRs), which are germ line-encoded pattern recognition receptors (PRRs)
that recognize “pathogen-associated molecular patterns” (PAMPs). 1,11 After
recognition of specific antigens, TLRs trigger NF- B, which then proceeds to
the nucleus and promotes the transcription and further synthesis of pro-inflammatory cytokines (TNF-a, IFN-g and IL-12) and chemokines.1 These responses also initiate the development of pathogen-specific, long-lasting adaptive immunity through B and T lymphocytes.11 While most reports on TLRs
have been focused on bacterial and fungal pathogens, studies confirm the importance of TLRs in the onset of leishmanial pathogenesis, susceptibility, and
resistance in mice and human disease models.12
Recent studies have demonstrated that TLRs, in particular TLR 2, 3, 4, 7,
8 and 9, play a major role in recognition of protozoan pathogens such as
Leishmania. However, limited information is available on Leishmania infection and TLRs. The majority of studies on TLRs have been performed in mice
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with cutaneous Leishmania species such as Leishmania major and only rarely
in humans or in macrophagic or dendritic cellular models.13-17 Deficient TLR
mice strains infected with L. major such as the TLR4-/-, and TLR9 -/- strains,
or studies performed with macrophages and dendritic cells derived from TLRdeficient mice could not efficiently control parasite replication and resolve cutaneous lesions. Therefore, most of the studies have shown that these TLRs
appear to up-regulate and to activate pro-inflammatory responses in mononuclear cells, which results in Leishmania parasite destruction and, therefore,
control of the infection.12 TLRs have not been studied in canine L. infantum
infection in detail. To date, only one recent study has studied TLRs in Leishmania-infected dogs. It has described higher values of TLR2 expression in
granulocytes and monocytes of infected Brazilian dogs, which were not infectious to sand flies by xenodiagnoses due to low Leishmania parasite burden in the skin.18
GENETIC CONTROL OF CANINE LEISHMANIOSIS
The host genetic background influences the progression of disease from
Leishmania infection. There is clinical and experimental evidence that suggest that some breed, for instance the Ibizian hound, appear to be more resistant to the disease.19
However, there have been very few genetic studies in dogs and these have
focused on a few candidate genes which have confirmed some genes previously found in mice and humans. For instance, a DLA class II DLA-DRB1
genotype, which is a dog MHC class II allele, has been linked to the risk of
susceptibility to leishmaniosis in an endemic area in Brazil.20 Moreover, a
study on the polymorphism of the Slc11a1 (Solute carrier family 11 member
a1) canine gene (former NRAMP1 gene), which encodes an iron transporter
protein involved in the control of intraphagosomal replication of parasites and
macrophage activation, has implied that susceptible dogs have polymorphism
and mutations in this gene.1, 21
Recently, it has been published a study on genome-wide association and
genomic selection approaches applied to a population-based case-control
dataset of 219 dogs from a single breed (Boxer).21 There was found that a substantial proportion of the genome was affecting the trait and that its heritability could be as high as 60%. Moreover, genomic selection estimated markers
in chromosome 4 (61.2–76.9 Mb) to have the greatest effect on the phenotype.
An important finding of that study was the significant predictive value from
using the genomic information (i.e the affection status was correctly predicted in 60% of dogs). 22
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BIBLIOGRAPHY
1. Tizard IR. 2013. Immunity to Parasites, pp 311-325. In Tizard IR (ed): Veterinary Immunology, 9th edition. Elesevier Saunders, St Louis, Missouri.
2. Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L. 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity
using PCR on several tissues and serology. Journal of Clinical Microbiology. 39: 560–
563.
3. Cabral M, O'Grady JE, Gomes S, Sousa JC, Thompson H, Alexander J. 1998: The immunology of canine leishmaniosis: strong evidence for a developing disease spectrum
from asymptomatic dogs. Veterinary Parasitology. 76(3): 173-180.
4. Baneth G, Solano-Gallego L. 2012. Canine leishmaniosis, pp 735-745. In Green CE (ed):
Infectious diseases of the dog and cat, 4th edition. 1. Elesevier Saunders, St Louis, Missouri.
5. Solano-Gallego L, Koutinas A, Miro G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P,
Oliva G, Baneth G. 2009. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veterinary Parasitology. 165(1-2): 1-18.
6. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. 2008. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends in Parasitology. 24(7): 324-330.
7. Reis AB, Giunchetti RC, Carrillo E, Martins-Filho OA, Moreno J. 2010. Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis. Trends in Parasitology. 26: 341-349.
8. Ribeiro-Gomes FL, Sacks D. 2012. The influence of early neutrophil-Leishmania interactions on the host immune response to infection. Frontiers in Cellular and Infections
Microbiology. 2:1-8.
9. Ribeiro-Gomes FL, Peters NC, Debrabant A, Sacks DL. 201. Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-Leishmania response.
PLoS Pathogens. 8: 1-12.
10. Lieke T, Nylen S, Eidsmo L, Schmetz C, Berg L, Akuffo H. 2011. The interplay between Leishmania promastigotes and human Natural Killer cells in vitro leads to direct lysis
of Leishmania by NK cells and modulation of NK cell activity by Leishmania promastigotes. Parasitology. 138:1898–1909.
11. Kawai T, Akira S. 2011. Toll-like receptors and their crosstalk with other innate receptors
in infection and immunity. Immunity. 34(5): 637-650.Kumar H, Kawai T, Akira S. 2011.
Pathogen recognition by the innate immune system. International reviews of immunology. 30(1): 16-34.
12. Tuon FF, Amato VS, Bacha HA, Almusawi T, Duarte MI, Amato Neto V. 2008. Toll-like
receptors and leishmaniasis. Infection and immunity. 76(3): 866-872.
13. Cezario GA, de Oliveira LR, Peresi E, Nicolete VC, Polettini J, de Lima CR, Gatto M,
Calvi SA. 2011. Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection. Mem Inst Oswaldo Cruz.
106(5): 573-583.
14. Zhang WW, Matlashewski G. 2008. Immunization with a Toll-like receptor 7 and/or 8
agonist vaccine adjuvant increases protective immunity against Leishmania major in
BALB/c mice. Infection and immunity. 76(8): 3777-3783.
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15. Flandin JF, Chano F, Descoteaux A. 2006. RNA interference reveals a role for TLR2 and
TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gammaprimed macrophages. European journal of immunology. 36(2): 411-420.
16. Tuon FF, Fernandes ER, Pagliari C, Duarte MI, Amato VS. 2010. The expression of TLR9
in human cutaneous leishmaniasis is associated with granuloma. Parasite immunology.
32(11-12): 769-772.
17. Gallego C, Golenbock D, Gomez MA, Saravia NG. 2011. Toll-like receptors participate
in macrophage activation and intracellular control of Leishmania (Viannia) panamensis.
Infection and immunity. 79(7): 2871-2879.
18. Amorim IF, Silva SM, Figueiredo MM, Moura EP, Castro RS, Lima TK, Gontijo Nde F,
Michalick MS, Gollob KJ, Tafuri WL. 2011. Toll receptors type-2 and CR3 expression of
canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in
visceral leishmaniasis. PLoS One, 6(11):e27679.
19. Solano-Gallego L, Llull J, Ramos G, Riera C, Arboix M, Alberola J, Ferrer L. 2000. The
Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Veterinary Parasitology. 90: 37–45.
20. Quinnell RJ, Kennedy LJ, Barnes A, Courtenay O, Dye C, Garcez LM, Shaw MA, Carter SD, Thomson W, Ollier WE. 2003. Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism. Immunogenetics. 55(1):23-8.
21. Sanchez-Robert E, Altet L, Utzet-Sadurni M, Giger U, Sanchez A, et al. 2008. Slc11a1
(formerly Nramp1) and susceptibility to canine visceral leishmaniasis. Veterinary Research. 39(3): 36.
22. Quilez J, Martínez V, Woolliams JA, Sanchez A, Pong-Wong R, Kennedy LJ, Quinnell
RJ, Ollier WE, Roura X, Ferrer L, Altet L, Francino O. 2012. Genetic control of canine
leishmaniasis: genome-wide association study and genomic selection analysis. PLoS
One. 7(4):e35349.
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Laura Ordeix
Esteve, Lda Vet, Dipl ECVD,
Barcellona (E)
Alessandra Fondati
Med Vet, PhD, Dipl ECVD, Roma (I)
Manifestazioni cutanee
nella Leishmaniosi canina
Venerdì, 8 Marzo 2013 - ore 14.10
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INTRODUZIONE
La Leishmaniosi canina, causata da Leishmania infantum, è spesso classificata come malattia viscerale in conformità con la classificazione della malattia causata da questo patogeno nell’uomo.1,2 Tuttavia, i cani hanno di solito sia il coinvolgimento viscerale sia il cutaneo.1 Infatti, in corso di Leishmaniosi canina, le lesioni cutanee sono la manifestazione clinica più comune, essendo riportate nell’81-89% dei cani malati.1,3
Nella Leishmaniosi canina sono state descritte diverse alterazioni cutanee,
sia dal punto di vista clinico sia istopatologico.3,4,5 In aree in cui la Leishmaniosi canina è endemica questo polimorfismo ha come conseguenza il fatto
che la malattia venga comunemente inclusa nelle diagnosi differenziali dei
problemi dermatologici.
Sia a fini diagnostici che terapeutici, è importante stabilire se le lesioni osservate siano attribuibili a Leishmania. Potrebbe infatti trattarsi di problemi
dermatologici i) imputabili a Leishmania in cani malati di leishmaniosi, ii)
non imputabili a Leishmania in cani infetti non malati, iii) non imputabili a
Leishmania in cani malati di leishmaniosi che hanno, oltre alla leishmaniosi,
un’altra malattia causa dei segni clinici cutanei.
Nell’uomo la leishmaniosi del sistema tegumentario ha uno spettro di manifestazioni cliniche ben definito (lesioni cutanee autolimitanti, leishmaniosi
cutanea, leishmaniosi disseminata, leishmaniosi delle mucose e leishmaniosi
cutanea diffusa).6
Studi svoltisi negli ultimi anni hanno migliorato la conoscenza di quanto i
fattori concernenti l’ospite ed il parassita contribuiscano alla patogenesi della
leishmaniosi cutanea umana.6 Questi studi indicano che nell’uomo l’infezione con ceppi diversi del parassita può portare a quadri clinici cutanei diversi.
Inoltre, per quanto riguarda l’ospite, la risposta immunitaria, sia acquisita sia
innata, può controllare l’infezione da Leishmania con conseguente ampio
spettro delle manifestazioni cliniche.6
Le cause della variabilità morfologica delle lesioni cutanee nel cane non
sono completamente conosciute, al pari di quanto accade nella leishmaniosi
umana. Tuttavia, s’ipotizza che la diversità della risposta immunitaria nei confronti di Leishmania infantum, probabilmente determinata geneticamente, sia
il fattore più importante a decidere i meccanismi patogenetici e quindi il tipo
di alterazioni dermatologiche osservate. 1,3,7
I problemi dermatologici clinici imputabili alla Leishmaniosi canina potrebbero essere suddivisi in tipici, più frequenti e/o più “caratteristici”, e
atipici, meno frequenti e/o più sovrapponibili a quelli causati da altre malattie. Entrambi possono essere osservati contemporaneamente nello stesso
paziente.
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QUADRI CLINICI CUTANEI TIPICI
I quadri clinici cutanei tipici includono la dermatite desquamativa, la dermatite ulcerativa in corrispondenza dei punti di appoggio e delle prominenze
ossee, e la dermatite papulare.
•
Dermatite desquamativa
Esiste una chiara evidenza che la dermatite desquamativa è il problema
dermatologico più comunemente riportato in corso di Leishmaniosi canina.4,8,9
La desquamazione è caratterizzata da scaglie secche, biancastre-madreperlacee (“amiantacee”), discretamente aderenti alla superficie cutanea, che
possono interessare solo la cute facciale, in particolare la regione perioculare,
il dorso del naso e i padiglioni auricolari, oppure estendersi alle estremità e al
tronco. Spesso la desquamazione è accompagnata da alopecia parziale e comunque di solito la precede.
Possono a volte osservarsi scaglie in forma di manicotti follicolari, sempre
di colore bianco-grigiastro. È stato ipotizzato che l’adenite sebacea piogranulomatosa osservata istologicamente sia causa, almeno in alcuni casi, delle scaglie e dei manicotti follicolari osservabili clinicamente.4,8 Il prurito è generalmente assente.8
L’adenite sebacea idiopatica è la diagnosi differenziale clinica principale.
Altre diagnosi differenziali da considerare includono la dermatofitosi, il linfoma cutaneo epiteliotropo, la seborrea secca, disordini di corneogenesi secondari a disturbi metabolici, ad esempio ipotiroidismo, e la cheyletiellosi,
specie in presenza di prurito.
All’esame dermatopatologico si può osservare una dermatite da perivascolare ad interstiziale nel derma superficiale e medio (perifollicolare),
con/senza distruzione delle ghiandole sebacee (adenite sebacea). Infatti,
l’adenite sebacea è stata osservata nella metà dei casi di dermatite desquamativa studiati.4,8
L’infiltrato infiammatorio dermico, composto principalmente da macrofagi, plasmacellule e linfociti, con occasionali neutrofili, eosinofili, mastociti e raramente con cellule giganti multinucleate, può assumere una distribuzione da multinodulare a diffusa e, meno frequentemente, estendersi al
tessuto adiposo sottocutaneo causando una pannicolite granulomatosa/piogranulomatosa.
Recentemente è stato suggerito che l’adenite sebacea non associata ad un
infiltrato nodulare-diffuso dermico probabilmente non è indicativa di leishmaniosi, anche nelle regioni in cui la malattia è endemica.10
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•
Dermatite ulcerativa in corrispondenza dei punti
di appoggio e delle prominenze ossee
Nella letteratura scientifica le dermatiti ulcerative da Leishmania sono frequentemente descritte come un’unica entità clinica.1,3,4,9 Tuttavia, le lesioni ulcerative-crostose possono derivare da diversi meccanismi patogenetici, essere
più o meno tipiche ed interessare sedi cutanee diverse.
Le lesioni ulcerative-crostose sono per frequenza il secondo tipo di lesioni cutanee osservate in corso di Leishmaniosi canina e sono state più frequentemente osservate in corrispondenza dei punti di appoggio, specialmente sulle articolazioni carpali e tarsali.4 Sono ulcere indolenti, superficiali e con
i bordi netti e rilevati, con un diametro variabile da 1 a 4 cm.4
Le diagnosi differenziali cliniche includono piodermite profonda localizzata, ulcere da decubito, neoplasie o granulomi infettivi da cause diverse dalla leishmaniosi, ad esempio una micosi profonda o una micobatteriosi.
L’esame dermatopatologico mostra il quadro istopatologico più comune
nella leishmaniosi cutanea, cioè una dermatite piogranulomatosa o granulomatosa da multinodulare a periannesiale a diffusa con ulcerazione.
•
Dermatite papulare
Si tratta di un problema dermatologico che può essere considerato molto
caratteristico e suggestivo di Leishmaniosi canina, quindi “tipico”, sebbene non
molto frequente.7,11
Clinicamente è caratterizzata dalla presenza di un numero variabile, di solito ridotto, di papule persistenti, rosa-giallastre, di consistenza aumentata, occasionalmente ombelicate, distribuite in aree pressoché glabre di cani a pelo corto (faccia interna dei padiglioni auricolari, addome ventrale, labbra, cute attorno al planum nasale).7,11 Le papule, che non tendono ad aumentare di volume
nel tempo, possono ulcerarsi nella parte centrale, assumendo quella morfologia
crateriforme riportata come tipica della leishmaniosi cutanea umana.
Come accennato in precedenza, in aree endemiche, è una manifestazione
cutanea molto suggestiva di leishmaniosi, tuttavia, nelle diagnosi differenziali andrebbero considerate reazioni a punture/morsi di artropodi non parassiti
(es. zanzare), lesioni della sindrome del piogranuloma-granuloma sterile o la
istiocitosi cutanea reattiva.
L’esame istopatologico delle biopsie cutanee rivela una dermatite piogranulomatosa da nodulare a diffusa con un basso numero di amastigoti intramacrofagici osservati frequentemente con l’uso di tecniche immunoistochimiche specifiche.7 Tuttavia, sono stati descritti alcuni casi in cui il numero di
amastigoti nelle lesioni era elevato.12
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Spesso i cani con dermatite papulare non hanno altre alterazioni clinicopatologiche ed il titolo anticorpale anti-Leishmania è negativo o debolmente
positivo. In alcuni casi è stata riportata una buona risposta immunitaria cellulo-mediata specifica nonché la risoluzione spontanea delle lesioni in 3-5 mesi.7,11 In conseguenza, è possibile che il numero variabili di amastigoti presenti
nelle lesioni descritto nei diversi studi, sia il risultato dell’età delle lesioni. Esseno progressivamente più basso in quelle lesioni più vecchie in animali in qui
la risposta inmunitaria ha controllato il numero di parassiti.
Infatti, in base alla recente classificazione clinica della leishmaniosi
cutanea, questa manifestazione viene considerata l’unica manifestazione
dermatologica ammessa nello stadio I, quindi associata ad una prognosi
favorevole.1
In base al quadro clinico-patologico-immunologico è stato ipotizzato che
si tratti di papule che insorgono nella sede di inoculazione e moltiplicazione
del parassita.7
QUADRI CLINICI CUTANEI ATIPICI
I quadri clinici cutanei di seguito riportati sono considerati da poco frequenti a rari rispetto ai precedenti e/o più facilmente attribuibili anche a cause diverse dalla leishmaniosi.
•
Dermatiti ulcerative atipiche
Le lesioni ulcerative-crostose meno frequenti possono presentarsi in corrispondenza di:
– Le giunzioni muco-cutanee (in particolare facciali)4
L’aspetto clinico, e a volte anche quello istopatologico, è sovrapponibile a
quanto osservabile nella piodermite-mucocutanea. Altre diagnosi differenziali da considerare in funzione dei dati del segnalamento e della storia clinica
sono il lupus eritematoso cutaneo, le reazioni avverse a farmaci o il linfoma
cutaneo epiteliotropo.
– Le sedi sottoposte a trauma prolungato (con soluzione di continuità della cute e difetto di cicatrizzazione)
Ad esempio dermatite da leccamento delle estremità o ferite chirurgiche.
In questi casi, si ipotizza che in cani infetti i macrofagi, richiamati dal normale processo infiammatorio-cicatriziale, veicolino amastigoti nella sede delle lesioni causando così l’aggravamento e la cronicizzazione del processo.
– La cute che ricopre le estremità (punta della coda, cuscinetti plantari,
margini dei padiglioni auricolari)4
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In quest’ultimo caso è probabile che la patogenesi delle ulcere sia, almeno in alcuni casi, legata a vasculite cutanea.13 Tuttavia, le presunte lesioni vascolari, anche a causa della loro sede, vengono raramente sottoposte a biopsia ed esame dermatopatologico, pertanto il danno vascolare non viene documentato frequentemente.
Talvolta, è possibile osservare onicomadesi come conseguenza del danno
vascolare accaduto nella matrice ungueale.
– Il planum nasale
Le alterazioni dermatologiche sono caratterizzate inizialmente da depigmentazione del planum e/o degli orifizi nasali, con posteriore sviluppo di erosioni, ulcere e croste.14 Le diagnosi differenziali da includere saranno diverse
a seconda della localizzazione delle lesioni. Nei casi in cui le erosioni e le ulcere sono ampie e colpiscono tutto il planum nasale, le diagnosi differenziali
cliniche sono: reazioni avverse a farmaci, inclusa la necrolisi epidermica tossica, il pemfigo volgare, il pemfigo paraneoplastico e le dermatosi bollose subepidermiche, autoimmuni o su base genetica. Se le lesioni sono distribuite
nella parte dorsale del planum nasale, la diagnosi differenziale principale è il
lupus eritematoso discoide. Infine, se le lesioni sono presenti solo nei solchi
alari (dorsali o ventrali), la diagnosi clinica differenziale più probabile è la
piodermite mucocutanea.14
All’esame dermatopatologico delle lesioni ulcerative nasali si può osservare una dermatite della giunzione dermo-epidermica con infiltrato linfoplasmacellulare-macrofagico con/senza neutrofili e con/senza danno dei cheratinociti
basali (vacuolizzazione e occasionale apoptosi) per le forme simil-lupus, o una
dermatite da multinodulare a diffusa piogranulomatosa con ulcerazione secondaria nelle altre forme ulcerative. Il numero di parassiti è variabile.
•
Onicopatie
Le onicopatie, specie la onicogrifosi, sembrano essere un problema dermatologico tipico della Leishmaniosi canina, poiché non sono comunemente
associate alla leishmaniosi umana.15,16 Nella Leishmaniosi canina la onicogrifosi viene riportata circa in un 24-90% dei casi, con una prevalenza superiore
a quella della paronichia (6-14%).15
Clinicamente la onicogrifosi è caratterizzata da ipertrofia e curvatura anomala degli artigli, in assenza di paronichia.15 Raramente si presenta come unico segno clinico della Leishmaniosi canina, infatti, la maggior parte dei cani
con onicogrifosi e leishmaniosi mostra altri problemi cutanei quali dermatite
desquamativa o ulcerativa.15
La diagnosi differenziale clinico-patologica più importante è la onichite
lupoide idiopatica. Di fatto, in assenza di altri segni cutanei suggestivi di lei66
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shmaniosi, la differenza può risultare difficile (vedi sotto). Altre diagnosi differenziali cliniche da considerare sono la onicomicosi e la onicogrifosi del cane geriatrico.
Le alterazioni istologiche della onicogrifosi associata alla leishmaniosi,
sebbene non specifiche, sono caratterizzate da una dermatite della giunzione
dermo-epidermica con infiltrato mononucleare (linfociti, plasmacellule ed
istiociti), vacuolizzazione dei cheratinociti basali, incontinenza pigmentaria e
separazione dermo-epidermica.15 Queste lesioni sono osservabili anche in corso di altre malattie, specie la onichite lupoide idiopatica. Inoltre, i segni istologici sono presenti anche nelle sezioni istologiche di cani con leishmaniosi
in assenza di onicogrifosi. È stata quindi ipotizzata la necessità di un periodo
prolungato, dopo l’inizio dell’infiammazione microscopica, perché la onicogrifosi diventi clinicamente evidente.15
La onicogrifosi non sembra essere il risultato della presenza di una elevata carica parassitaria nel derma. La difficoltà di dimostrare una presenza “significativa” di parassiti nell’infiltrato attraverso esami immunoistochimici
specifici o tecniche di biologia molecolare,15 assieme alla non specificità dei
segni istopatologici, fa sì che, almeno in alcuni casi, la imputazione delle lesioni a Leishmania sia difficile.
•
Dermatite nodulare cutanea e muco-cutanea
La dermatite nodulare cutanea e muco-cutanea è piuttosto infrequente nella Leishmaniosi canina.4 La sua prevalenza è stata riportata fra il 2 e il 17%
in una serie di casi.5 È stata suggerita una certa predisposizione per il cane di
razza Boxer.17
Si tratta di noduli cutanei, singoli o multipli, di solito localizzati in aree coperte di pelo, a differenza delle papule sopradescritte, di cui è poco probabile
che rappresentino l’evoluzione. La cute sovrastante i noduli ha per lo più
aspetto normale ma è possibile che si ulceri. In alcuni casi sono state riportate lesioni papulari-nodulari delle giunzioni muco-cutanee e delle mucose
esterne (orale, genitale…).18,19 I noduli, sia cutanei che muco-cutanei e delle
mucose esterne, vengono normalmente attribuiti alla disseminazione per via
linfo-ematogena del parassita. Tuttavia, è stato ipotizzato che flebotomi contenenti Leishmania possano essere “schiacciati” dal cane nel cavo orale ed essere così causa in alcuni casi di lesioni papulari sulla superficie linguale.20
Le diagnosi differenziali sono processi neoplastici, particolarmente il linfoma cutaneo epiteliotropo per la forma orale, calcinosi circumscripta, amiloidosi, granulomi eosinofilici, granulomi infettivi da cause diverse dalla leishmaniosi, ad esempio una micosi profonda o una micobatteriosi, e dermatiti
granulomatose sterili.
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L’esame dermatopatologico evidenzia una dermatite granulomatosa o piogranulomatosa diffusa, talvolta con cellule giganti e con un numero variabile,
ma frequentemente elevato, di amastigoti.5,17
•
Dermatite pustolosa sterile
È stata occasionalmente riportata una dermatite pustolosa superficiale sterile.4,9 È considerata una manifestazione clinica poco comune e difficile da
imputare a Leishmania, almeno dagli studi riportati in precedenza.
Clinicamente è caratterizzata dalla presenza di pustole intatte assieme a
collaretti epidermici presenti nella superficie ventrale del tronco. Il prurito è
variabile.4 Istologicamente si osserva una dermatite pustolosa neutrofilica
subcorneale-intraepidermica con discreta spongiosi ed in assenza di acantolisi. Gli amastigoti sono presenti nel derma in numero variabile, ma non all’interno delle pustole.4,9
Dal punto di vista diagnostico è importante stabilire se le lesioni pustolose osservate in un cane con leishmaniosi sono attribuibili a Leishmania. Potrebbe infatti trattarsi di i) cani malati di leishmaniosi con una piodermite superficiale, ii) cani malati di leishmaniosi con una dermatite pustolosa superficiale immunomediata (es. pemfigo foliaceo, dermatite pustolosa eosinofilica,
dermatosi neutrofilica sterile, dermatite pustolosa superficiale da reazione avversa a farmaci),21 iii) cani malati di leishmaniosi e dermatite pustolosa sterile imputabile a Leishmania. A complicare il problema deve essere anche presa in considerazione la possibilità che una dermatite pustolosa sterile, scatenata da una qualunque causa, possa infettarsi secondariamente da cocchi.
Per confermare l’associazione causa (Leishmania) - effetto (dermatite pustolosa superficiale) in questa manifestazione clinica è necessario i) eseguire
esami citologici e batteriologici del contenuto di pustole intatte e confermare
in questo modo la sterilità delle lesioni, ii) eseguire esami dermatopatologici
di pustole intatte ed escludere in questo modo alterazioni dermatopatologiche
suggestive di malattie pustolose sterili immunomediate, iii) eseguire esami
immunoistochimici specifici e determinare in questo modo la presenza di Leishmania nell’infiltrato dermico ed infine, iv) valutare la risposta clinica favorevole alla sola terapia anti-Leishmania. È essenziale inoltre valutare le condizioni clinico-patologiche generali del paziente oltreché il titolo anticorpale
anti-Leishmania specifico.
•
Dermatite desquamativa naso-digitale
Un’altra forma dermatologica atipica della Leishmaniosi canina è la dermatite desquamativa naso-digitale.4 Quando presente, di solito è associata ad
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altri segni clinici, quali ad esempio la dermatite desquamativa in aree non glabre. È stato infatti suggerito che la dermatite desquamativa naso-digitale sia
una forma localizzata della stessa manifestazione in forma generalizzata.4
Le lesioni cliniche osservate sono scaglie secche e aderenti occasionalmente accompagnate da ragadi lineari. Le lesioni sul naso sono più abbondanti sul planum dorsale e quelle presenti sui cuscinetti plantari possono essere causa di disagio nell’andatura.4,14
Tra le diagnosi differenziali si includono la dermatite che risponde allo
zinco, l’ipercheratosi idiopatica, più frequente nei cani anziani, la paracheratosi ereditaria del labrador retriever e, raramente, il cimurro.14
Il quadro istopatologico non è ben definito. A causa della diagnosi di leishmaniosi attraverso la presenza di altri segni clinici e patologici diagnostici
e della loro sede, queste lesioni vengono raramente sottoposte a biopsia.
•
Alopecia multifocale
In rari casi si osservano aree di alopecia multifocale di aspetto cicatriziale riconducibili ad una dermatopatia ischemica, da confermare con l’esame dermatopatologico. In questi casi la presenza di Leishmania intralesionale potrebbe
essere difficile da dimostrare anche con tecniche molto sensibili (es. PCR).
PUNTI CHIAVE:
1. La maggior parte dei cani malati di leishmaniosi mostra lesioni cutanee.
2. Le manifestazioni dermatologiche cliniche sono polimorfe.
3. Alcune manifestazioni cliniche dermatologiche sono tipiche (frequenti e/o
molto suggestive della malattia) mentre altre sono atipiche (molto meno
frequenti e/o sovrapponibili a malattie diverse alla leishmaniosi).
4. Il quadro istologico tipico, osservato nella maggior parte delle manifestazioni dermatologiche cliniche, è caratterizzato da una reazione infiammatoria caratterizzata dalla presenza di macrofagi, linfociti e plasmacellule.
5. Alcune manifestazioni cliniche dermatologiche (dermatite ulcerativa della
cute che ricopre le estremità, dermatite ulcerativa del planum nasale simillupus, onicopatie) presentano lesioni istologiche atipiche.
6. La correlazione tra il quadro clinico, quello istopatologico e il numero (ridotto-elevato) di parassiti intralesionali non è costante.
7. Quando le lesioni istologiche sono suggestive di leishmaniosi ma nei campioni colorati di routine o con Giemsa non si osservano parassiti (a causa
del numero ridotto) è necessario confermarne/escluderne la presenza mediante tecniche immunoistochimiche e/o biomolecolari (PCR).
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BIBLIOGRAFIA
1. Baneth G, Solano-Gallego L. 2012. Canine leishmaniosis, pp 735-745. In Greene CE
(ed): Infectious diseases of the dog and cat, 4th edition. 1. Elsevier Saunders, St Louis,
Missouri.
2. McCall LI, Zhang WW, Matlashewski G. 2013. Determinants for the Development of Visceral Leishmaniasis Disease. PLOS Pathogens. 9: 1: e1003053.
3. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P,
Oliva G, Baneth G. 2009. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Veterinary Parasitology. 165: 1–18.
4. Koutinas AF, Scott DW, Kantos V, Lekkas S. 1992. Skin lesions in canine leishmaniasis
(Kala-Azar): A Clinical and Histopathological Study on 22 Spontaneous Cases in Greece. Veterinary Dermatology. 3: 121-130.
5. Blavier A, Keroack S, Denerolle Ph, Goy-Thollot I, Chabanne L, Cadoré JL, Bourdoiseau G. 2001. Atypical forms of canine leishmaniosis. Veterinary Journal. 162: 108-120.
6. Carvalho LP, Passos S, Schriefer A, Carvalho EM. 2012. Protective and pathologic immune responses in human tegumentary leishmaniasis. Frontier in Immunology. 3: Article 301.
7. Ordeix L, Solano-Gallego L, Fondevila D, Ferrer L, Fondati A. 2005. Papular dermatitis
due to Leishmania spp. infection in dogs with parasite-specific cellular immune responses. Veterinary Dermatology. 16: 187-191.
8. Papadogiannakis EI, Koutinas AF, Saridomichelakis MN, Vlemmasb J, Lekkasb S, Karamerisc A, Fytianou A. 2005. Cellular immunophenotyping of exfoliative dermatitis in canine leishmaniosis (Leishmania infantum). Veterinary Immunology and Immunopathology. 104: 227-237.
9. Ferrer L, Rabanal R, Fondevila D, Ramos JA, Domingo M. 1988. Skin lesions in canine
leishmaniasis. Journal of Small Animal Practice. 29: 381-388.
10. Bardagí M, Fondevila D, Zanna G, Ferrer L. 2010. Histopathological differences between canine idiopathic sebaceous adenitis and canine leishmaniosis with sebaceous adenitis.Veterinary Dermatology. 21: 159-165.
11. Bottero E, Poggi M, Viglione M. 2006. Lesioni papulari indotte da Leishmania spp. in 8
cani giovani. Veterinaria, 20: 33-36.
12. Noli C, Cornegliani L. 2006. Leishmaniosi bottoniforme. Descrizione di cinque casi
italiani e confronto con la letteratura veterinaria. Quaderni di dermatología. 11(1):
23-26.
13. Pumarola M, Brevik L, Badiola J, Vargas A, Domingo M, Ferrer L. 1991. Canine leishmaniasis associated with systemic vasculitis in two dogs. Journal of Comparative Pathology. 105(3):279-86.
14. Fondati A, Ordeix L. 2012. Malattie del planum nasale, pp 170-175. In De Lorenzi D
(ed): Malattie dell’apparato respiratorio nel cane e nel gatto. Elsevier, Milano, Italia.
15. Koutinas AF, Carlotti DN, Koutinas C, Papadogiannakis EI, Spanakos GK, Saridomichelakis MN. 2010. Claw histopathology and parasitic load in natural cases of canine
leishmaniosis associated with Leishmania infantum. Veterinary Dermatology. 21(6):
572-7.
16. Mahmood K. 2007. An unusual paronychia. Diagnosis: paronychial cutaneous leishmaniasis. Clinical Experimental Dermatology. 32(5):611-2.
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17. Ferrer L, Fondevila D, Marco A, Pumarola M. 1990. Atypical nodular leishmaniasis in
two dogs. Veterinary Record. 126: 90.
18. Font A, Roura X, Fondevila D, Closa JM, Mascort J, Ferrer L. 1996. Canine mucosal leishmaniasis. Journal of the American Animal Hospital Association. 32(2):131-7.
19. Viegas C, Requicha J, Albuquerque C, Sargo T, Machado J, Dias I, Pires MA, Campino
L, Cardoso L. 2012. Tongue nodules in canine leishmaniosis — a case report. Parasites
& Vectors 2012, 5:120.
20. Foglia Manzillo V, Pagano A, Paciello O, Di Muccio T, Gradoni L, Oliva G. 2005. Papular-like glossitis in a dog with leishmaniosis, Veterinary Record 156: 213-215.
21. Ginel PJ, Mozos E, Fernández A, Martinez A, Molleda JM. 1993. Canine pemphigus foliaceus associated with leishmaniasis. Veterinary Record. 133(21):526-7.
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Domenico Otranto
DVM, Dr Ric, Dipl EVPC, FRES, Bari, Italy
DVM, MSc, DSc, PhD, FRES, Dipl EVPC, Brazil
New canine vector-borne
diseases in the old and new worlds
Saturday, March 9th 2013 - ore 5.35 pm
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CANINE VECTOR-BORNE DISEASES IN THE OLD
AND NEW WORLDS
Canine vector-borne diseases (CVBDs) are a group of diseases caused by
a range of pathogens that are transmitted by arthropod vectors, such as ticks,
mosquitoes and phlebotomine sand flies. Babesiosis, dirofilariosis, ehrlichiosis and leishmaniasis are examples of CVBDs that cause relevant morbidity
and mortality in dogs around the world. In the past decades, the risk of vector-borne pathogen transmission in dogs has increased considerably and this
situation has been attributed to changes in vector ecology due to recent climate changes. Moreover, changes in human behaviour, increased animal trafficking and movements from south to north have favoured the introduction of
vector-borne pathogens into non-endemic areas.
Besides the well-known CVBDs (e.g., leishmaniosis, dirofilariosis, ehrlichiosis and babesiosis) other diseases have emerged recently in the Old and
the New Words, thus posing a further challenge to veterinarians and eventually medical physicians, as some emerging vector-borne pathogens of dogs
have the potential to infect humans.
The burden of vector-borne diseases in a given country may depend on a
number of factors, including socioeconomic status (e.g., income levels, education levels, and health-related behaviours) and the efficiency of the veterinary medical infrastructure. In developed countries, dog care and welfare are
usually maintained at high standards, at least before the economic crisis of the
past decade occurred. In addition, the number of dogs travelling with their
owners has increased considerably, ultimately leading to a higher risk of introduction of exotic CVBDs. Meanwhile, the importation and relocation of infected animals from an endemic to a non-endemic area may be extremely critical as demonstrated for dogs infected by Dirofilaria immitis from the areas
stricken by Hurricane Katrina (e.g., New Orleans, Louisiana) into other nonendemic areas of the United States. In the same way, the existence of public
kennels hosting stray dogs regardless their health condition, in areas where
CVBDs are endemic (e.g., Italy and Spain), contributes to the maintenance of
the endemicity of many vector-borne diseases, both in suburban and urban areas. This is the case of dirofilariosis and leishmaniasis that are increasingly
being detected in some European (e.g., Italy) and South America countries
(e.g., Brazil and Argentina).
In developing countries vector-borne diseases of human concern (e.g.,
malaria, lymphatic filariasis, and dengue fever), along with those affecting
livestock production, constitute a high priority whereas CVBDs remain considerably less regarded, despite the high number of stray dogs and the high risk
of zoonotic transmission by arthropod vectors living in the same microenvi73
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ronments. Moreover, many dog owners cannot handle the costs of preventative
tools (e.g., insecticides and vaccines) and thus the risk of vector-borne
pathogen infection in privately owned dogs might be as high as in stray dogs.
NEW VECTOR-BORNE PATHOGENS FOUND IN DOGS
Besides the well-known Dirofilaria immits, canine filarial infestations are
due to nematodes whose adults colonize predominantly subcutaneous tissues,
muscular fasciae, retropharyngeal and axillary lymphatics (genera Acanthocheilonema, Brugia, Cercopithifilaria and Dirofilaria), and ocular tissues
(Onchocerca lupi). Much attention is currently being given to the clinical impact of these parasites in the veterinary practice, with the aim of improving
treatment and control. The life cycle of most filarioids of dogs either those
vectored by mosquitoes, back flies or ticks is little studied and, for most of
them, remains largely unknown. Recently, other onchocercids infesting dogs
have gained the interest of the scientific community and many aspects of their
life history have been investigated.
Cercopithifilaria spp.
Cercopithifilaria adults are usually beneath cutaneous tissues and are
transmitted by hard ticks, their microfilariae being always in the dermis. The
first species known to infest dogs, namely Cercopithifilaria grassii, was described more than one century ago and it is characterized by typical “gigantesche” (from Italian, giant) microfilariae. Recently, microfilariae of Cercopithifilaria sp. (herein after referred as Cercopithifilaria sp. I) have been morphologically and molecular characterized from a dog from Sicily (Italy). The
occurrence of this nematode overlapped with the distribution of its vector (the
brown dog tick, Rhipicephalus sanguineus) and it was retrieved with prevalence rates reaching up to 21.6% in dogs from Spain, Greece and southern
Italy. Interestingly, the wide distribution of Cercopithifilaria sp. I in canine
populations paralleled the high nucleotide variation among mitochondrial cytochrome c oxidase subunit 1 sequences of examined nematodes (n=2,287)
from skin samples and ticks, with up to 16 haplotypes characterized to date.
Larvae of Cercopithifilaria sp. I are distributed unevenly in the superficial
dermal tissues of infested dogs, being mostly present on the skin of head, ears
and neck regions, which are also among the most common attachment sites of
R. sanguineus. Recent histological evidence indicated that microfilariae of
Cercopithifilaria sp. I may induce erythematous and papular dermatitis in infested dogs. Furthermore, recent morphological and molecular evidence indi74
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cated that at least three Cercopithifilaria spp. are present in the Mediterranean
area (i.e., Cercopithifilaria sp. I, C. grassii, and a yet undescribed microfilaria, Cercopithifilaria sp. II).
Onchocerca lupi
An emerging filarioid nematode infesting the eyes of dogs is O. lupi, a parasite described from periocular tissues of a Caucasian wolf (Canis lupus) in
Georgia. In dogs, cases of ocular onchocerciasis have been reported in southern (Greece, Portugal) and Central Europe (Germany, Hungary, Portugal,
Switzerland). Since the first case of canine onchocercosis recorded in western
United States, O. lupi was demonstrated as the causative agent in two cats and
in dogs. This species has been also identified as a causative agent of human
infestation in Turkey and Tunisia, in patients who exhibited clinical features
similar to those of canine infestation. Undoubtedly, further research on the biology, clinical impact and immune response elicited by these parasites are advocated in order to gain more information on their actual impact on veterinary
medicine and, for O. lupi, its potential as a zoonotic agent.
Rangelia vitallii
An enigmatic parasite found in dogs in the New World is Rangelia vitalii.
This protozoon belongs to the phylum Apicomplexa and order Piroplasmida,
being thus closely related to Babesia species. It is typically found in young
dogs living in rural and suburban areas in Brazil. Though to be transmitted by
ticks (e.g., Amblyomma aureolatum and Rhipicephalus sanguineus), R. vitalii
remains a little known parasite. In a recent study, seven young female dogs
were experimentally infected with R. vitalii and monitored by blood smear examinations, which revealed intra-erythrocytic forms 5 days post-infection,
with a peak of parasitemia between 9 and 11 days post-infection. Protozoa
were seen inside the leukocytes at days 17, 19 and 21 post-infection. At the
day 20 post-infection, signs such as lethargy, fever and anorexia were recorded. Other alterations recorded were decreased packed cell volume and moderate anaemia. Importantly, a single dose of diminazene aceturate showed a
100% efficacy in cleaning R. vitalii from blood of infected dogs.
Trypanosoma caninum
A new species of trypanosome was recently isolated from intact skin of
dogs from south-eastern Brazil. The actual pathogenic role of this interesting
protozoon remains doubtful and little is known regarding its life cycle. Stud75
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ies have indicated that T. caninum is present in some areas where canine leishmaniasis is endemic and it has been shown that infection by T. caninum may
lead to a misdiagnosis of canine visceral leishmaniasis due to potential crossreactivity occurring with standard serological tools.
FINAL CONSIDERATIONS
The changing epidemiology of CVBDs poses an additional challenge to
veterinary practitioners and medical physicians working in both Old and New
Worlds. The emergence or re-emergence of CVBDs may be driven by several factors linked to vectors, pathogens and susceptible dog populations as well
as to owners’ attitude towards disease prevention. The use of molecular biology in research has enable the delineation of new species such as T. caninum,
but the use of molecular tools in the daily diagnosis routine of veterinarians
working in developing countries is still far from reality. Nonetheless, additional knowledge on the species of vector-borne pathogens infecting dogs in
both Old and New Worlds is needed to improve our understanding of the related risks the microorganisms may pose to dogs and also to humans.
Domenico Otranto
Department of Veterinary Medicine, University of Bari,
Str Prov per Casamassima km 3, 70010
Department of Veterinary Medicine, University of Bari, Str Prov per Casamassima km 3,
70010 Valenzano/BA, Italy; 2Department of Immunology, Aggeu Magalhães
Research Institute, Av Prof Moraes Rego s/n, 50670420 Recife/PE, Brazil
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Domenico Otranto
DVM, Dr Ric, Dipl EVPC, FRES, Bari, Italy
Canine vector borne disease
of human concern
Sunday, March 10th 2013, 11.20 am
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Vector-borne diseases represent about 17% of the burden of all infectious
diseases. Their importance is not only related to the economic losses to the
livestock industry but there has been increasing concern about the global
spread of parasitic arthropods and vectored pathogens between pets, specifically dogs, and humans. Indeed, the social role played by dogs in developed
countries and their increasingly close relationship with humans in both urban
and rural areas pose new threats for public health. Canine vector-borne diseases (CVBDs) are caused by a group of pathogens and some of them are of
major zoonotic concern. Dogs are exposed to a high number of parasitic
arthropods (e.g. ticks, fleas, mosquitoes, and phlebotomine sand flies), which
act as efficient vectors of a large number of bacteria, viruses, protozoa, and
helminths affecting also human beings.
In developing countries, vector-borne diseases of human concern (e.g.
malaria and leishmaniasis), along with those affecting livestock health and
production, constitute the highest priority issue for public health and veterinary services. Consequently, CVBDs remain a considerably lower priority,
despite the high number of stray dogs and the high risk of zoonotic transmission. Nonetheless, in both developing and developed countries, data on the
distribution of arthropods and CVBDs are often scant, most likely as a result
of lack of surveillance at local and regional levels. Consequently, the development of new and ultimately endemic foci of CVBDs in non-endemic areas
may occur in the absence of efficient veterinary and public health surveillance
networks, possibly leading into the rapid spread of the infection within human
and animal populations.
TICK-BORNE DISEASES
Tick-borne diseases (TBDs) affect human and animal populations in terms
of morbidity and mortality. Moreover, reported cases of TBDs, including
Rocky Mountain spotted fever, Lyme borreliosis, and tick-borne encephalitis,
are on the rise. Humans may be accidental hosts for some tick species (e.g.,
Ixodes ricinus and Rhipicephalus sanguineus) especially persons who work
or live in close contact with infested domestic animals. Information of TBDs
has greatly increased in recent years and many important zoonotic TBDs,
such as anaplasmosis, babesiosis, ehrlichiosis, and Lyme borreliosis, are
nowadays well known to medical and veterinary medicine. A number of new
species, strains or genetic variants of microorganisms are being detected in
ticks around the world, and the list of actual or potential tick-borne pathogens
continues to increase. Some of these agents, such as Rickettsia slovaca, Rickettsia parkeri and Rickettsia massiliae were identified in ticks many decades
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before these were associated with human diseases. Other pathogens, including the flaviviruses have recently been implicated as causes of human disease
in new geographical regions. However, the pathogenic potential of many bacterial species detected recently in ticks around the world remains mostly unknown and the continuous discovery of new microorganisms may impact on
human and veterinary medicines. Tick-borne pathogens traditionally associated with disease in domestic animals may eventually emerge as human
pathogens. For example, Ehrlichia canis has been implicated in a few human
cases in Venezuela.
LEISHMANIOSIS
The leishmanioses are caused by Leishmania protozoa and vectored by
phlebotomine sand flies. Most Leishmania spp. are zoonotic, involving reservoir hosts such as rodents, marsupials, edentates, monkeys, domestic dogs
and wild canids worldwide. Human leishmaniosis is caused by zoonotic
Leishmania spp., and only a few are anthroponotic (i.e., transmitted directly
from human to human via phlebotomine sand flies). Dogs are the major reservoir for canine and human L. infantum infection worldwide. In Europe, the
domestic dog is the only reservoir host of major veterinary and human importance although infection in other animal species (e.g., cats, wild canids,
horses, and hares) has also been reported. Natural transmission is usually by
the bite of an infected phlebotomine sand fly whereas the epidemiological importance of other way of transmission (venereal, transplacentary, blood transfusion) remains unclear.
It has been estimated that 2.5 million dogs in Europe are infected with L.
infantum, and infection is spreading into areas previously regarded as non endemic of central and north Europe. The infection rate of canine population in
the Mediterranean basin varies according to the test used (i.e., from 5 to 30%
by serology, up to 70% by molecular tools) and to geographical area. Indeed,
the distribution of this disease is mostly related to the occurrence of the proper species of phlebotomine sand fly vectors.
After malaria and lymphatic filariasis, human leishmaniasis represents the
third most important vector-borne disease in many tropical and sub-tropical
regions of the Old and New World. According to the species of Leishmania
involved, human leishmaniasis may occur in a visceral form (VL), involving
internal organs, and in cutaneous or mucocutaneous forms (CL), which affect
the skin or mucocutaneous junctions and may heal spontaneously. Leishmaniasis is endemic in 88 countries, with more than 350 million people at risk
and an estimated incidence is 2 million new cases per year (0.5 million VL
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and 1.5 million CL). In Europe long-term endemism has been demonstrated
for VL and CL by L. infantum and anthroponotic CL by L. tropica (sporadically in Greece). VL primarily affects children as well as an increasing rate of
immunocompromised and immunosuppressed adult individuals, such as HIVinfected and patients under any immunosuppressive therapies.
DIROFILARIOSIS AND OTHER ZOONOTIC HELMINTHS
Dirofilariosis caused by Dirofilaria spp. is a widespread helminthic disease transmitted by several mosquito species. Dirofilaria immitis, the agent of
cardio-pulmonary disease in dogs, is considered the main agent of human
dirofilariosis in the Americas. Dirofilaria repens, which is usually localized in
the subcutaneous tissue of dogs, is the main agent of human dirofilariosis in
the Old Word. Dirofilaria immitis and D. repens infect mainly dogs and cats,
but also other hosts such as wild carnivores and humans. Dirofilaria spp. develop throughout five larval stages within the intermediate vector mosquito
host (from embryo to infective L3 larva), and in the definitive vertebrate host
(from L3 to the adult stage). The intermediate hosts are mosquitoes (e.g.,
Culex, Aedes, Ochlerotatus, Anopheles, Armigeres and Mansonia), although
Aedes vexans, Culex pipiens and Aedes albopictus are mainly implicated vectors in Europe. Dirofilaria repens is able to grow under laboratory conditions
in the same mosquito species as D. immitis and at the same temperature and
humidity as D. immitis. A recent re-examination of 28 cases of human dirofilariosis reported in the last 30 years in the Old Word indicated that D. repens
was, indeed, the most prevalent species causing human infestation
Another neglected filarioid infesting the eyes of dogs and cats is Onchocerca lupi, a parasite causing ocular onchocerciasis in southern (Greece,
Portugal) and Central Europe (Germany, Hungary, Portugal, Switzerland).
Since the first case of canine onchocercosis recorded in western United
States, O. lupi was also detected in two cats. This species has been also identified in humans in Turkey and Tunisia. Undoubtedly, further research on the
biology, clinical impact and immune response elicited by these parasites are
advocated in order to gain more information on their actual impact on veterinary medicine and, for O. lupi, its potential as a zoonotic agent.
Thelazia callipaeda, has been reported infesting the eyes of humans as
well as domestic (dogs and cats) and wild carnivores, including foxes,
wolves, beech martens and wild cats. In the past decade, T. callipaeda was
proved to be widespread among dogs and cats from northern (Aosta valley) to
southern Italy (Basilicata region) with a prevalence as high as 60% in dogs
from some municipalities. Autochthonous cases of thelaziosis in dogs and
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cats have been described in south-western France, Switzerland, Portugal and
Spain. The first four cases of human thelaziosis in Europe have been diagnosed in the Piedmont region of Italy in patients coming from an area of
north-western Italy and south-eastern France.
The distribution of this nematode is closely associated to its vector, Phortica variegata and the various hosts.
CONCLUSIONS
Despite recent advancements in scientific knowledge related to CVBDs of
zoonotic concern, their management and control is still impaired by a number
of critical issues. Without a doubt a One Health approach to CVBD, will increase the chances for a successful management of these diseases in both developed and developing countries.
Department of Veterinary Medicine, University of Bari
Str Prov per Casamassima km 3, 70010 Valenzano/BA, Italy
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Maria Grazia Pennisi
Med Vet, Specialista in Microbiologia
Applicata, Messina
Laia Solano-Gallego
DVM, PhD, Dipl ECVCP,
Barcellona (E)
Leishmaniosi felina:
tra certezze e dubbi
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INTRODUZIONE
La leishmaniosi nel gatto suscita molteplici interessi scientifici. Infatti oltre all’interesse clinico per chiarire vari aspetti della malattia esiste un interesse di tipo epidemiologico che mira a valutare l’entità del fenomeno (prevalenza dell’infezione) e il possibile ruolo di serbatoio del gatto. Questa eventualità avrebbe importanti ripercussioni di sanità pubblica per le leishmaniosi
zoonosiche se si pensa all’elevato numero di gatti che vivono vicino all’uomo
come animali da compagnia o anche come randagi.
Questo numero supera infatti da tempo in Europa e nei paesi industrializzati quello dei cani (FEDIAF Facts & Figures 2010). Francia, Italia e Spagna
sono in particolare nazioni in cui la leishmaniosi zoonosica da L. infantum è
endemica in vaste aree del paese e il numero di gatti di proprietà risulta particolarmente elevato.
L’INFEZIONE NEL GATTO
Il gatto è suscettibile all’infezione naturale ad opera di diverse specie del
genere Leishmania come risulta da studi svolti in varie parti del mondo, usualmente in aree endemiche per l’infezione umana o canina (Pennisi, 2010). Anche l’infezione sperimentale ha confermato questo dato (Kirkpatrick et al.,
1984; Simoes-Mattos et al., 2005).
Il parassita è stato isolato da animali con malattia spontanea e identificato
anche a livello di zimodema mediante analisi elettroforetica degli isoenzimi,
oppure l’identificazione è stata confermata con metodiche molecolari. In Europa tutti i casi identificati sono stati attribuiti a L.(chagasi) infantum per la
quale il cane è considerato il più importante serbatoio. In alcuni di essi si è
provveduto alla caratterizzazione isoenzimatica riscontrando per lo più lo zimodema MON1, quello più comune nella leishmaniosi viscerale zoonosica,
ma in un caso è stato osservato il MON72 (secondo in ordine di prevalenza in
Italia) e in un altro il MON 201 (ritrovato in Italia in un paziente umano con
coinfezione da HIV) (Ozon et al., 1998; Gramiccia et al., 2005; Maroli et al.,
2006). Sempre L. (chagasi) infantum è stata identificata nel gatto in Iran (Hatam et al., 2010) e in Brasile (Savani et al., 2004; Cohelo et al., 2010a; Vides
et al., 2011). Nel Nuovo Mondo altre specie sono state identificate nel gatto
in Brasile (L. (Viannia) braziliensis, L. amazonensis) (Schubach et al., 2004;
de Souza et al., 2005), Venezuela (L. venezuelensis) (Bonfante-Garrido et al.,
2001), Guiana Francese (L. (Viannia) braziliensis) (Rougeron et al., 2011) e
Texas-USA (L. mexicana)(Craig et al., 1986; Barnes et al., 1993; Trainor et
al. 2010). In Messico è stato di recente ipotizzato che il gatto possa anche ri83
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sultare contemporaneamente infetto da più specie di Leishmania (L. infantum,
L. braziliensis e L. mexicana) (Longoni et al., 2012). Possiamo quindi affermare che nel gatto la leishmaniosi è causata dalle stesse specie che infettano
il cane e l’uomo. Ciò significa che è importante comprendere appieno il ruolo epidemiologico del gatto per capire se può fungere da serbatoio primario
(al pari del cane) o secondario per una zoonosi inserita a pieno titolo nella lista delle “Neglected Tropical Diseases” per l’impatto negativo che ha sulla salute umana nelle aree più povere del pianeta. Nel momento in cui grandi sforzi vengono compiuti per la prevenzione integrata della Leishmaniosi canina
con presidi repellenti e con vaccini, l’individuazione di eventuali altri serbatoi animali si fa sempre più importante.
Un dato sperimentale significativo, seppure ottenuto con una prova svolta
su un solo gatto, è che Phlebotomus perniciosus, principale vettore di L. infantum in Italia, si infetta compiendo il pasto di sangue su un gatto ammalato
con infezione cronica (Maroli et al., 2006). Non sappiamo però se, come accade nel cane, anche i gatti sieropositivi con infezione subclinica sono infettanti per il vettore. Se così fosse, prevalenza e incidenza dell’infezione nella
popolazione felina sarebbero parametri epidemiologici importanti come succede per quelli dell’infezione canina. Lo stesso dato è stato ottenuto in Brasile con Lutzomia longipalpis, il più importante vettore della malattia in Brasile, per cui questo quesito si ripropone anche nel Nuovo Mondo (Magno da
Silva et al., 2010).
È singolare infine che in Tailandia, dove si sospetta che una nuova specie
di Leishmania sia l’agente di alcuni casi di leishmaniosi viscerale autoctona,
le indagini sierologiche svolte su animali domestici (cani, gatti, capre, conigli, ratti, oche, anatre, polli) presenti nelle vicinanze dell’abitazione di un paziente hanno dato esito positivo solo con alcuni sieri felini (Sukmee et al.,
2008).
ASPETTI DIAGNOSTICI
Tecniche sierologiche e/o biomolecolari sono state utilizzate negli ultimi
decenni per indagini epidemiologiche nel gatto. Tra le tecniche sierologiche
sono state utilizzate Immunofluorescenza indiretta (IFI), tecniche immunoenzimatiche (ELISA), agglutinazione diretta (DAT) o Western Blott (WB). Nonostante siano state pubblicate decine di studi, che forniscono prevalenze
comprese fra 0 e 68%, manca ancora la validazione di un test per la ricerca di
anticorpi anti-Leishmania nel gatto.
Nelle indagini in cui è stata impiegato l’IFI, sono stati usati cut-off molto
diversi (da 1:2 a 1:100) fissati spesso in maniera arbitraria e non sempre vie84
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ne riportata la tipologia dei sieri di controllo utilizzati: dovrebbero essere della stessa specie con controllo positivo ottenuto da gatti con infezione confermata. Sieri felini ottenuti da gatti che vivono in zone non endemiche, affetti
da patologie diverse, devono essere testati per evidenziare eventuali cross-reazioni che possono dare falsi positivi soprattutto a basso titolo. In questo modo è possibile fissare il cut off in base al quale discriminare i campioni positivi dai negativi.
Anche per quanto riguarda la tecnica ELISA per fissare il cut off sono stati impiegati criteri eterogenei e sono stati utilizzati antigeni diversi (l’antigene ricombinante K39, il recettore fucosio-mannosio o antigene grezzo) che
offrono risultati discordanti fra loro (da Silveira Neto et al., 2011). La stessa
discordanza si osserva anche fra ELISA e IFI, come del resto è possibile anche nel cane e l’ELISA ha in genere una maggiore sensibilità (Cohelo et al.,
2011; da Silveira Neto et al., 2011; Sobrinho et al., 2012).
In aree dell’America latina in cui è endemico anche Trypanosoma cruzi è
stato utilizzato con successo come antigene per ELISA o WB una superossido-dismutasi escreta che risulta essere un marker specifico per differenziare
le due specie (Longoni et al., 2012). Allo stesso modo sono stati validati metodi sierologici che consentono di diagnosticare anche nel gatto in maniera
specifica l’infezione da T. cruzi (Sartor et al., 2005). Non esiste invece crossreazione fra anticorpi anti-Leishmania e anti-Toxoplasma, come del resto accade anche nel cane (Nasereddin et al., 2008; Sherry et al., 2011; da Silveira
Neto, 2011; Coehlo et al., 2011; Sobrinho et al., 2012).
Il DNA del parassita è stato ricercato per la prima volta nel sangue di gatti nel 2000 (Pennisi et al., 2000) e negli anni successivi altri studi analoghi
hanno riscontrato prevalenze comprese fra 0 e 60.6%. In rari casi l’indagine è
stata svolta su grandi campioni impiegando matrici considerate più sensibili
nel cane come il midollo osseo, la milza, il linfonodo o i tamponi congiuntivali. Il midollo osseo è però risultato negativo in uno studio su 95 gatti, anche
in quelli di loro in cui la PCR dal sangue era positiva o erano presenti anticorpi (Veronesi et al., 2010).
Del resto, anche in ambito citologico il midollo osseo sembra avere nel
gatto una minore sensibilità diagnostica rispetto a linfonodo e milza (Coehlo
et al., 2010b; Sobrinho et al., 2012). Per contro linfonodo e tampone congiuntivale hanno dimostrato una sensibilità maggiore del sangue, come del resto accade anche nel cane (Pennisi et al. 2012). La possibilità di utilizzare i
tamponi congiuntivali per l’esecuzione della PCR risulta particolarmente interessante per gli studi epidemiologici perché mette a disposizione una metodica sensibile effettuabile con prelievo rapido, non invasivo ed economico che
ben si presta per studi biomolecolari su larga scala ancora poco praticati (Pennisi et al., 2012).
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In tutti gli studi in cui sono state applicate contestualmente metodiche bimolecolari e sierologiche è emersa una discordanza fra test diretti e indiretti
(Pennisi et al., 2000; Martin –Sanchez et al. 2007; Ayllon et al., 2008; Sherry
et al., 2011; Maia et al., 2010; Millan et al., 2011; Pennisi et al., 2012; Sobrinho et al., 2012). Ciò significa che gatti infetti possono essere sieronegativi. Come succede nel cane, la positività immunologica (anticorpale e/o cellulare) e/o biomolecolare in soggetti subclinici potrebbe testimoniare una resistenza del gatto che può condurre a risoluzione spontanea, ma anche rappresentare solo una fase di passaggio nello stato di equilibrio immunitario dell’animale infetto del quale non è facile predire la dinamica né in termini temporali né prognostici.
Tuttavia, la risposta cellulomediata non è stata valutata nel gatto. Malattie
intercorrenti o terapie immunosoppressive sono due potenziali fattori esogeni
che possono interferire con le difese immunitarie e portare l’ospite da una
condizione di infetto subclinico a quella di ammalato.
FATTORI DI RISCHIO
Vari elementi sono stati presi in considerazione come fattore di rischio
per l’infezione: ambientali (altitudine, contesto rurale/urbano) stile di vita
(all’aperto/al chiuso, contatto con altri gatti o con altre specie animali), stagione, dati segnaletici (razza, sesso, età), coinfezioni (Feline Immunodeficiency Virus (FIV), Feline Leukemia Virus (FeLV), Coronavirus, Toxoplasma), stato di salute. Nasereddin (2012) ha riscontrato nel territorio di Gerusalemme una prevalenza più elevata in gatti che vivono in ambito collinare (altitudine ≥ 760 m). Riguardo ai dati segnaletici, due studi hanno segnalato prevalenze maggiori nei maschi (Cardoso et al., 2010; Sobrinho et al.,
2012) o in gatti di età > 2-3 anni (Cardoso et al., 2010; Ayllon et al., 2011;
Pennisi et al., 2012).
Un fattore per il quale si sono avuti risultati a volte contrastanti riguarda
il ruolo delle infezioni da retrovirus. Per quanto riguarda FeLV, solo uno
studio riporta una associazione significativa fra infezione da FeLV e da Leishmania (Sherry et al., 2011), mentre in un altro studio nei gatti con FeLV
la siero prevalenza è invece significativamente più bassa (Martin-Sanchez et
al., 2007). Riguardo a FIV, alcuni studi non rilevano alcuna associazione
con l’infezione da Leishmania (Vita et al, 2005; Martin-Sanchez et al.,
2007; Solano-Gallego et al., 2007), in altri casi è invece presente (Ayllon et
al., 2011; Pennisi et al., 2012; Sobrinho et al., 2012). È un aspetto quest’ultimo che si ricollega ai numerosi casi di malattia descritti in gatti FIV positivi oppure FIV e FeLV positivi (Pennisi, 2010).
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STUDI LONGITUDINALI
Pochi sono i dati ottenuti da studi longitudinali compiuti sul gatto che
possono dare un’idea più precisa sul possibile ruolo di serbatoio del gatto.
In uno studio su 15 gatti (7 positivi e 8 negativi per anticorpi anti-FIV) con
reattività anticorpale per Leishmania variabile (titolo compreso fra <20 e
1280) durante un monitoraggio compreso fra 3 e 23 mesi, il titolo anticorpale non ha avuto modificazioni superiori a due diluizioni e la PCR dal sangue è risultata positiva in 50 campioni su 72 complessivamente testati, senza differenze significative in base all’infezione da FIV (Pennisi et al., 2000).
Questo dato depone a favore della possibilità di una infezione cronica da
Leishmania nel gatto. Un caso paradigmatico in questo senso è quello di un
gatto che, positivo per anticorpi anti-Leishmania con titolo compreso fra 40
e 80 per 6 anni e PCR positiva dal sangue, ha presentato dopo ripetuti trattamenti immunosoppressivi per una grave gengivostomatite cronica, linfoadenomegalia generalizzata associata all’infezione da Leishmania confermata mediante coltura e PCR (Maroli et al., 2006).
FELIDI SELVATICI
Un altro dato analogo riguarda l’infezione e la malattia nei felidi selvatici.
In passato, uno studio svolto in Italia su un piccolo gruppo di leonesse viventi in un parco zoo in area endemica aveva dato esito negativo sia alla ricerca
di anticorpi specifici che alla PCR su sangue, linfonodo e biopsia di cute (Laricchiuta et al., 2006). Recentemente invece a Montpelier (Francia) in una leonessa, nata in Marocco ma ospitata da 7 anni presso il locale parco zoo, è stata diagnosticata una infezione da L. infantum associata a un quadro clinico
cronico compatibile con leishmaniosi: cachessia, colite emorragica, epistassi,
zoppia e dermatite ulcerativa sui cuscinetti palmari e plantari, anemia moderata, leucocitosi, ipercolesterolemia e ipoalbuminemia (Libert et al., 2012).
L’animale era negativo per FIV, FeLV e positivo alla PCR sul sangue per L.
infantum e Mycoplasma candidatus haemominutum e aveva un titolo IFAT
anti-Leishmania di 80. Altre quattro leonesse conviventi e clinicamente sane
sono state sottoposte a prelievi che hanno dato esito positivo in una di esse: in
questo soggetto il titolo IFAT era 320 e PCR, coltura e citologia dal midollo
osseo hanno dato esito positivo. È singolare che grazie a campioni di siero di
questi due soggetti stoccati in sieroteca è stato dimostrato che la sieropositività era presente da almeno 5 anni nella leonessa ammalata e da 3 anni in
quella con infezione subclinica.
CONCLUSIONE
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La leishmaniosi felina è una patologia emergente in cui i casi di malattia
descritti sono ancora pochi in relazione al numero di gatti che vivono in aree
endemiche e alla presunta prevalenza dell’infezione riportata dalle indagini
epidemiologiche. Questo dato può significare che il gatto è un animale relativamente resistente o che dobbiamo affinare le nostre capacità diagnostiche
andando oltre la semplice applicazione di metodiche in uso per il cane.
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Indirizzo per la corrispondenza:
Maria Grazia Pennisi - Ordinario di Clinica Medica Veterinaria
Dipartimento di Scienze Veterinarie - Università di Messina, Italia
Laia Solano-Gallego - Dep. Medicina i Cirurgia Animal
Facultat de Veterinària - Universitat Autonoma Barcelona, Spagna
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Xavier Roura
DVM, PhD, Dipl ECVIM-CA
Barcelona, Spain
Usual and unusual clinical
pictures other than the cutaneous
signs associated with canine
leishmaniasis
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The most important characteristic of canine leishmaniasis is its extraordinary clinical polymorphism. Canine leishmaniasis is a systemic or visceral
disease; this means that numerous systems or organs can be affected and that
from a practical point of view it must be included among the majority of differential diagnoses. As the clinical signs of canine leishmaniasis are not
pathognomonic, a thorough evaluation of the clinical signs, history and physical examination are very important in order to confirm the possible correlation between Leishmania infection and the clinical signs in the dog.
It should for example not be forgotten that dogs of all breeds can be affected by leishmaniasis, although some, such as the German Shepherd and the
Boxer, seem to be more susceptible. A greater predisposition for the disease
is apparently present also in males, as described in humans and in hamsters.
In addition, Leishmaniasis has a bimodal distribution, with a peak of dogs affected under the age of 3 and a second peak between 8 and 10 years of age. It
is also very important to understand if the dog lives or has been in areas endemic for canine leishmaniasis, if it may have been exposed to the vectors
which transmit the disease (sandflies) or if it has undergone treatments which
may have altered the functioning of its immune system. The clinical history is
completed with a description of the clinical signs reported by the owner and
which may be compatible with canine leishmaniasis.
The clinical signs present with leishmaniasis are caused by two main
pathogenetic principles: a nonsupporative granulomatous inflammation within the sites of multiplication of the parasite and the deposit of immune complexes in distinct anatomical sites. The first mechanism is responsible for
clinical manifestations involving the skin, liver, intestine, eyes, kidneys,
bones and mucosae; the second is mostly the cause of renal, ocular and vascular lesions. In canine leishmaniasis the pathogenetic mechanisms underlying the presence of anaemia and of haemostatic disorders are still not known
with certainty. Most authors believe that the main cause of these alterations is
the presence of immune-mediated processes together with the fact that leishmaniasis is a chronic disease. Amyloid deposits in different organs have been
reported in some cases, however the clinical importance of this finding does
not seem particularly relevant.
Cutaneous ulcerations are the most frequent clinical sign, being present in
approximately 80% of affected dogs. Lymphadenopathy, present in around
70-80% of the dogs, and general symptoms (fever, apathy, weight-loss and
muscle atrophy), present in around 40-60%, are also very frequent. A broad
range of other clinical signs may also be present, such as renal and/or ocular
signs, splenomegaly, hepatomegaly, pain, lameness, diarrhoea, epistaxis,
onycogryphosis, onychorrexis, fever, jaundice, syncope or cough; the incidence of all these signs varies between 1% and 20%, depending on the author.
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The most frequent renal alteration is glomerulonephritis; in the dog, such condition is characterized by proteinuria, which may then evolve into a nephrotic syndrome, which may in some cases end up in a case of renal failure. Classical gastrointestinal clinical signs are diarrhoea, with or without melena, and
vomiting; both are related to colitis, duodenitis or secondary to renal disorders. Chronic hepatitis is an occasional finding. Epystaxis, present in approximately 10% of cases, is one of the clinical signs which is most difficult to explain, as its pathogenesis includes vasculitis, thrombocytopenia, coagulopathies,
hyperviscosity and ulcerative inflammation of the nasal mucosa.
Basic laboratory tests include a complete blood count, a serum biochemistry profile, a serum proteinogram and urinalysis. In the presence of canine
leishmaniasis these tests may allow to detect one or more alterations caused
by the disease. However in most cases, although helpful, alterations in the
clinical assays are not pathognomonic of canine leishmaniasis. The most frequent analytical alterations are hypergammaglobulinaemia (present in around
80% of cases), anaemia (in around 60%) and hypoalbuminaemia (in around
40%). The anaemia is of the non-regenerative type and depends on the fact
that leishmaniasis is a chronic disease; in the presence of a positive ANA and
Coombs test, the search for other possible explanations is recommended. The
haemogram usually shows signs of thrombocytopenia (60%) and, depending
on the case, leucocytosis or leucopenia. The hypergammaglobulinaemia is
usually polyclonal, although some monoclonal-type cases have been reported. The hypoalbuminaemia is compensatory for the hypergammaglobulinaemia as well as the consequence of glomerulonephritis. Other possible alterations are an increased ALT, γGT, urea or creatinine and the presence of
proteinuria or haematuria.
Based on all this information, the Veterinarian must decide on whether
leishmaniasis should be included or not within the list of differential diagnoses. In any case, if the clinical signs allow to include leishmaniasis within
the differential diagnoses, additional laboratory investigations in order to confirm or exclude its presence are recommended.
Hospital Clínic Veterinari, Universitat Autònoma de Barcelona
E-mail: xavier.roura@uab.cat
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Xavier Roura
Barcelona, Spain
Old and new strategies
of Leishmaniosis treatment
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Drug treatment of leishmaniasis in dogs is a challenge for veterinary practitioners. Because of its complex pathogenesis, leishmaniasis may manifest
with various clinical signs, ranging from mild and nonspecific to those reflecting severe involvement of several organs. The immune response plays an
important role in the development, outcome, and response to treatment of
Leishmania infection in dogs. All known anti-Leishmania drugs used in dogs
can lead to temporary or permanent remission of clinical signs, but usually
none are sufficient to eliminate the infection.
In dogs, the objectives of anti-Leishmania treatment are typically to induce
a general reduction of the parasite load, to treat organ damage caused by the
parasite, to restore efficient immune responses, to stabilize a drug-induced
clinical improvement, and to treat clinical relapse.
Therapeutic options and choice of drug regimens for dogs with leishmaniasis should be considered in light of the different clinical forms of the disease.
As described in the previous presentation, dogs can be classified in 4 clinical
stages of leishmaniasis: A, exposed dogs; B, infected dogs; C, sick dogs (dogs
with patent leishmaniasis); and D, severely sick dogs. However, for therapeutic purposes, an additional stage E is suggested, which includes dogs unresponsive to recommended treatment (stage E-a) or dogs that have a relapse of
clinical disease soon after completion of recommended treatment (stage E-b).
The most widely used treatment protocol for dogs with leishmaniasis is the
combination of meglumine antimoniate and allopurinol. This combination
may be administered to all dogs in stage B, C, or D, with meglumine antimoniate given at 100 mg/kg, SC, once a day for 4 weeks and allopurinol at 10
mg/kg, PO, every 12 hours for at least 6 months. The dosage of meglumine
antimoniate can be divided in 2 equal doses of 50 mg/kg (q 12 h) or administered for a period ranging from 4 to 8 weeks. In most dogs in stages B and C,
this protocol, if correctly applied, should result in a clinical cure that is stable
for more than 1 year. Adverse effects are reported with either drug of the combination. In addition, the protocol should result in a considerable decrease in
parasite load for several months, which is necessary for reducing transmission
of the parasite to sand flies.
For dogs in stage D with a severe clinical form of the disease, the aforementioned treatment protocol should yield a good chance of improvement but
may not result in a clinical cure. In stage-D dogs, particular if renal insufficiency is present, the need for ancillary treatments and prognosis are strictly
dependent on pre-existing clinical conditions.
Unresponsiveness to a previous course of recommended treatment (stage
E-a), occurrence of relapse soon after treatment (stage E-b), development of
severe adverse effects, poor owner compliance with drug administration
(problems in the application or cost), veterinary clinician preferences and,
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non–governmental approval or availability of drugs in a given country could
be reasons for choosing a treatment protocol other than meglumine antimoniate–allopurinol.
Choice of an alternative anti-Leishmania drug or drug combination should
be based on the following criteria: anti-Leishmania efficacy as established via
clinical trials; few potential adverse effects; and owner compliance with administration. The few alternative regimens that fulfil the aforementioned requirements include miltefosine given for 28 days (2 mg/kg, PO q 24 h) administered in combination with allopurinol, for at least 6 months (10 mg/kg,
PO, q 12 h); or allopurinol, administered alone for at least 6 months.
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Xavier Roura
Barcelona, Spain
New strategies
for Leishmaniosis prevention
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In a broad sense, the term prevention includes the application of measures
intended to avoid instances of infection by a pathogen or the pathologic outcome of such instances. Leishmaniasis control strategies have varied little for
decades, but in recent years there have been advances in the prevention of the
disease. Advances in prevention include evidence confirming that the incidence of leishmaniasis, in both humans and dogs, can be reduced.
As a brief summary, three canine leishmaniasis prevention strategies have
shown to be effective and are used ever more commonly: (1) in the dog, the regular use of topical insecticides – repellents (pyrethroids), (2) vaccination and (3)
oral treatment with domperidone. It is however necessary to understand that the
protection of each single dog, although high, is not 100% guaranteed with any
of these methods. The preventive efficacy of pyrethroids is of 84-96% in the individual animal and of 100% at population-level; the preventive efficacy of vaccination is of 70% in the individual animal, and of 80% using domperidone. The
various existing preventive strategies can be combined, in order to increase their
efficacy, however no data is available confirming that this approach increases the
degree of protection compared to their use alone. In areas where leishmaniasis
is endemic the use of topical insecticides with a proven efficacy against sandflies is recommended. Pyrethroids, and specifically deltamethrin and permethrin, are the agents for which a greater efficacy has been shown.
Deltamethrin collars (efficacy starts 7 days after application and is guaranteed for approximately 5 months) must be worn at the beginning of the sandfly
season; permethrin pipettes/spot-on solutions (efficacy starts 2 days after application and is guaranteed for approximately 3-4 weeks) must be applied from
April to November. Permetrhin sprays may also be used (efficacy starts immediately after application and is guaranteed for approximately 3 weeks), however
this last formulation is not commercialized in all countries. According to available data, leishmaniasis vaccination (3 vaccinations, each 3 weeks apart, followed by an annual booster) or domperidone (0,5 mg/Kg SID P.O. for 30 days
with repetition of the treatment 2 or 3 times a year depending on the geographical area) do not eradicate the infection; they do however induce a strong cellular immune response which prevents the development of the disease. Furthermore, in dogs already affected by the disease these interventions may result in
an effective immunotherapy, helping in the control of the disease by the immune
system. Preventive measures are recommended in any healthy dog which is to
visit areas in which leishmaniasis is endemic; the same is true for any dog which
is either infected or sick with leishmaniasis, whether under treatment or not, as
an effective strategy to reduce the risk of infecting both humans and dogs.
Hospital Clínic Veterinari, Universitat Autònoma de Barcelona - E-mail: xavier.roura@uab.cat
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Xavier Roura
Barcelona, Spain
Leishmaniosis and co-infections
with other canine
vector-borne diseases
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Canine leishmaniosis is endemic in the Mediterranean basin, where the
prevalence of infection in dogs can be as high as of 67% of selected populations. Currently, there is limited information regarding the prevalence of many
vector borne infections in dogs residing in specific geographical areas of Europe where leishmaniosis is an endemic infectious disease.
Some of the manifestations of leishmaniosis are similar or identical to
clinical signs, and hematological and biochemical abnormalities reported in
dogs with other vector borne infections. Clinical features of leishmaniosis can
vary widely in sick dogs or in people as a consequence of organism or host
specific factors. It is also possible that diversity of disease manifestations associated with leishmaniosis in some individuals is related to co-infection with
other vector borne organisms.
In recent years an increasing number of publications have described simultaneous infection with another vector borne pathogen in dogs that had a
classical presentation for leishmaniosis. A few published examples include
co-infections with Leishmania and Neospora, Ehrlichia, Hepatozoon, Bartonella, Babesia or Dirofilaria. For some organisms, the association between
the two infectious entities occurs with a greater frequency than would be expected based upon the incidence of each respective infection. For instance, an
epidemiological study performed in Italy found Neospora caninum seroreactivity as a major risk factor for Leishmania infantum seroreactivity. Co-infections should be expected in dogs living in areas that are highly endemic for
several vector borne organisms, in dogs that are maintained predominantly
outdoors (enhanced vector transmission) and in dogs that are not routinely
treated with acaracides or other ectoparacidacides.
Although co-infections could occur merely as a function of life style, it is
also possible that one or more vector borne organisms suppress the host immune response to other organisms that would typically be eliminated immunologically. Concomitant disease with other vector-borne organisms may
affect the severity and manifestation of the disease and, chronic immunosupression induced by different pathogens may predispose dogs to secondary infections. Has been described that several infections can promote an abnormal
response or suppression of the immune system, this has been described in
leishmaniosis, bartonellosis and ehrlichiosis. As examples, infection with
Leishmania infantum can induce suppression of the immune system or promote an abnormal response, and result in an imbalance between Th-1 and Th2 responses; or infection with Bartonella vinsonii (berkhoffii) can result in
CD8+ lymphocytopenia, impaired monocytic phagocytosis and impaired antigen presentation to helper T cells.
However, the majority of published studies could not demonstrate a statistical association between co-infections and presence of more quantity or
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severity of clinical signs. All the studies found similar percentage of co-infections either in healthy dogs, unhealthy dogs, Leishmania infected dogs or
dogs with patent leishmaniosis. Therefore, it is possible that co-infection occurs merely as a function of lifestyle, as all the vectors implicated in the transmission of these pathogens are common in an outdoor environment. But, because in few studies have been reported an increased severity of clinical signs
with co-infection, for example when Leishmania infantum and filariae co-infection is present, future seroepidemiological studies incorporating a larger
population of dogs or molecular based studies will be required to elucidate the
immunopathogenic role of other vector borne organisms in dogs living in
Leishmania infantum endemic regions.
In the other hand, Wolbachia is an intracellular, Gram-negative bacteria
belonging to the order Rickettsiales. Like other filarial species, Dirofilaria immitis harbours the endosymbiont bacterium Wolbachia, which plays a key role
in the biological processes of filaria-like embryogenesis and moulting. Recent studies have found that Wolbachia is involved in immune polarization
during infection, suggesting that the role of Wolbachia in the host response to
filarial infection may be due to the inflammatory/immunomodulatory activity
of Wolbachia-associated proteins. These studies showed a lower prevalence of
Wolbachia in microfilaremic dogs infected with Leishmania infantum. Some
antigens of Wolbachia could contribute to the stimulation of the Th1-type immune response, as has been previously described, and this fact could be advantageous for some dogs infected with Leishmania infantum.
Therefore, regional veterinarians, who are very familiar with leishmaniosis, should consider the possibility of co-infection. Sequential or concurrent infection with vector borne organisms could induce deleterious alterations in the dog’s immune response, leading to atypical or unusual disease
manifestations.
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Xavier Roura
Barcelona, Spain
Mycoplasmosis
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Feline and canine haemoplasmas are extremely small gram-negative bacteria localized on the surface of red blood cells which may be the cause of disorders ranging from a subclinical infection to a more or less severe haemolytic anaemia. In spite of this, at least in Spain, their existence is often neglected by clinical veterinarians and haemoplasmas are not always included within the differential diagnoses of various clinical cases; this is probably due to
the fact that their clinical and pathological role is not yet clear and that in reality their prevalence in many geographical areas has not yet been established.
The three species of haemoplasma which may infect the cat (based on the
16S rRNA gene sequence) are: Mycoplasma haemofelis previously known as
Haemobartonella felis, ‘Candidatus Mycoplasma haemominutum’ previously
known as Haemobartonella felis, and the more recently discovered ‘Candidatus Mycoplasma turicensis’.
Dogs can be infected by two additional species: Mycoplasma haemocanis
and ‘Candidatus Mycoplasma haematoparvum’. A fourth haemoplasma,
‘Candidatus Mycoplasma haematoparvum-like’, has been identified in the
cat, however this latter one has not yet been totally characterized.
‘Candidatus Mycoplasma haemominutum’ is the most frequently encountered feline mycoplasma, with a prevalence of positivity in cats ranging
between 7% and 32% in various studies. Mycoplasma haemofelis, ‘Candidatus Mycoplasma turicensis’ and “Candidatus Mycoplasma haematoparvumlike” are less frequent and their prevalence varies, respectively, depending on
the study, between 1.4% and 6.4%, between 1.3% and 26% and at 3,75%. The
prevalence of canine leishmaniasis is also extremely variable, and depends on
the geographical area being assessed; for M. haemocanis the incidence varies
between 0,5% and 45% while for ‘Candidatus M. haematoparvum’ it varies
between 0% and 33%.
The clinical manifestations of the different species of haemoplasma vary
from a subclinical infection to a severe haemolytic disease. The most frequent
clinical signs, in both the cat and the dog, include dehydration, inappetence
(at times aberrant appetite), anorexia, debility, lethargy, weight-loss, depression, fever which at times may be intermittent (especially during the acute
stages of the infection), tachycardia/tachypnoea, hepatosplenomegaly, pale
mucosae and jaundice. The most frequent laboratory abnormalities are
hypochromic and macrocytic regenerative anaemia, hyperbilirubinaemia and,
in many cases, a positive Coombs test. In the dog and cat, a clear association
between the presence of anaemia and haemoplasma infection is apparently
not present. However various studies suggest a certain correlation between the
presence of anaemia in the cat and the type of haemoplasma present. These
studies suggest that M. haemofelis is often the cause of anaemia and clinical
signs, while ‘Candidatus M. haemominutum’ is the cause of a practically clin103
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ically inapparent infection with minimal CBC alterations, unless in the presence of a co-infection with feline leukaemia virus. On the contrary, co-infection with haemoplasma and feline immunodeficiency virus does not seem to
worsen the clinical cases of anaemia. In the dog, mycoplasma-induced
haemolytic anaemia is associated with splenectomy, immunosuppression
(treatments with chemotherapy or glucocorticoids) or with co-infections.
Blood diagnostics have a low sensitivity and specificity. In view of this,
in the presence of haemolytic anaemia in a cat a molecular assay is recommended, in order to identify the distinct types of feline haemoplasmas. It
should however not be forgotten that carriers without clinical signs are a
common finding, and hence a positive PCR test is not conclusive and a global assessment of all the laboratory results and of the clinical signs is therefore necessary.
In the cat, tetracyclines such as doxycycline (10 mg/kg/day P.O. for 4-8
weeks), and fluoroquinolones such as enrofloxacin (5 mg/kg/day P.O. for one
month), marbofloxacin (2 mg/kg/day P.O. for one month) or pradofloxacin (5
mg/kg/day P.O. for one month) are effective in the treatment of haemoplasma
infection. In the dog, to date no controlled studies on the treatment of haemoplasmosis have been done, however tetracyclines and fluoroquinolones have
proven effective in the treatment of individual clinical cases.
In the presence of mycoplasmas, in both the cat and the dog, ectoparasite
control (fleas and ticks) may be one of the most effective modalities to prevent the infection and the disease.
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COMUNICAZIONI BREVI
SHORT COMMUNICATIONS
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SEROLOGICAL AND MOLECULAR EVALUATION
OF LEISHMANIA INFANTUM INFECTION IN STRAY CATS
IN A NON-ENDEMIC AREA IN NORTHERN ITALY
E. Spada, DVM, PhD, Researcher 1, A. Della Pepa, DVM 1,
A. Migliazzo, DVM, PhD 2, G. Bagnagatti De Giorgi, DVM 1,
R. Perego, DVM, PhD 1, D. Proverbio, DVM, PhD, Professor 1
1
Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e
la Sicurezza Alimentare, Università degli Studi di Milano, Milan, Italy
2
Centro di Referenza Nazionale per le Leishmaniosi,Istituto Zooprofilattico
Sperimentale della Sicilia, Palermo, Italy
Tipologia: Ricerca Originale
Area di interesse: Medicina interna
Purpose of the work. To assess the prevalence of leishmaniosis in a large representative sample of stray cats from this non-endemic area, and to analyze
the results according to clinical, laboratory and infectious data.
Materials and used methods. Blood samples were collected from 233 European shorthair stray cats from urban colonies in Milan, northern Italy,
during a trap-neuter-release (TNR) program. The following data were
recorded: sex (n=233), age (n=233), body condition score (n=215), area of
colony of provenance i.e. one of the seven municipalities of Milan (n=233),
health status based on physical examination (n=233), and dermatological
evaluation (n=121), complete blood cell count (n=127), antibodies to FIV
relative to the gp40 and p24 FIV antigens, the FeLV p27 antigen (n=137),
and Toxoplasma gondii IgG antibodies (n=79). The presence of anti-L. infantum antibodies was measured by an indirect immunofluorescence antibody test (IFAT) performed according to the recommendations of OIE using
MHOM/IT/80/IPT1 as a whole-parasite antigen fixed on multispot slides
and fluorescently-labeled anti-feline gamma as conjugate. L. infantum DNA
was amplified from 200 µl of whole blood by real-time PCR.
The target for amplification was a 116-bp fragment in the constant region
of the kDNA minicircle.
The primers used were QLK2-UP 5’-GGCGTTCTGCGAAAACCG-3’ and
QLK2-DOWN 5’-AAAATGGCATTTTCGGGCC-3’; the TaqMan probes
were Q Leish Probe 2 and 5’-FAM TGGGTGCAGAAATCCCGTTCA-3’Black Hole. Factors associated with the L. infantum positivity were considered statistically significant when P<0.05.
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Outcomes. The serology test for L. infantum showed that 25.3% (59/233) of
the cats had L. infantum seroreactivity, 38 (16.3%) had antibody titers of
1:40, 15 (6.4%) had titers of 1:80 and 6 (2.6%) had antibody titers of 1:160.
All blood samples tested using real time PCR were negative for the presence
of L. infantum DNA. Only FIV seropositive status was statistically associated
with seroreactivity to L. infantum (multivariate logistic regression: P=0.0098
and OR = 7.34 (95%CI=1.96 to 27.59).
Conclusions. This study is the first epidemiological investigation of feline
Leishmania infection in the metropolitan area of Milan, which is a non-endemic area for leishmaniosis. Our results were surprising, since no autochthonous canine cases of leishmaniosis have ever been reported in this region in northern Italy. In countries in southern Europe where leishmaniasis is
endemic, serological investigations performed in feline populations using different techniques have revealed prevalence rates that range from less than 1%
to more than 60%.1 As here, previous epidemiological studies have used IFAT
to detect antibodies to Leishmania spp. in cats.1,2,3,4
None of the peripheral blood samples we examined using real-time PCR
were positive for parasite DNA. PCR has been used previously by others,
either alone or in combination with serology, as in our study, to assess the
prevalence of feline Leishmania infection. Blood is not the best specimen
for PCR diagnosis of leishmaniasis. Specifically, PCR performed on canine
blood has lower sensitivity, specificity and positive and negative predictive
values compared to PCR performed on canine lymph node aspirates,5 and
this may be true for samples from cats as well. However, blood sampling is
less invasive and is easy to perform, particularly for epidemiological studies involving numerous subjects, as in our survey. In addition, in many previous studies, L. infantum DNA was found in whole blood samples from
cats.1,2,3,4
Our survey may be an overestimation due to the possibility of IFAT crossreactivity between L. infantum and other pathogens, possible on some serologic tests, especially those that use a whole-parasite antigen, as we did
here. This has been demonstrated in dogs in that IFAT cross-reactivity has
been reported for L. infantum and Trypanosoma cruzi, Leishmania braziliensis and Ehrlichia canis infection.6
There was no significant correlation between T. gondii-positivity and L. infantum-positivity in our study. This may suggest a lack of cross-reactivity with
Toxoplasma parasites.
Additional studies that include parasite isolation are needed to clarify our
findings on feline leishmaniasis in this geographic area.
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Bibliography
1. Pennisi MG. A high prevalence of feline leishmaniasis in southern Italy. In: KillickKendrick, R. (Ed), canine Leishmaniasis: Moving Towards a Solution. Intervet International, Boxmeer, 2002, pp 39-48.
2. Tabar MD, Altet L, Francino O, et al. Vector-borne infections in cats: Molecular study in
Barcelona area (Spain). Vet Parasitol 2008; 151: 332–336.
3. Pennisi MG, Lupo T, Malara D, et al. Serological and molecular prevalence of Leishmania
infantum infection in cats from southern Italy. Clinical/research abstracts accepted for presentation at ISFM Congress 2012. J Feline Med Surg 2012; 14: 650-658
4. Martín-Sánchez J, Acedo C, Muñoz-Pérez M, et al. Infection by Leishmania infantum in
cats: epidemiological study in Spain. Vet Parasitol 2007; 145: 267-273.
5. Reale S, Maxia L, Vitale F, et al. Detection of Leishmania infantum in Dogs by PCR with
Lymph Node Aspirates and Blood. J Clin Microbiol 1999; 37: 2931-2935.
6. De Castro Ferreira E, de Lana M, Carneiro M, et al. Comparison of serological assays for
the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet Parasitol 2007; 146: 235-241.
Corresponding Address:
Dott.ssa Eva Spada, Via Celoria, 10, 20133 Milano (MI), Italia
Cell. 349/6766663 - E-mail: eva.spada@unimi.it
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RETRIEVAL OF LEISHMANIAL DNA IN A COMMERCIALLY
AVAILABLE LiESP/QA-21 VACCINE
T. Furlanello, Dr Med Vet, PhD, ECVCP Dip 1,2,
M. Trotta, Dr Med Vet, PhD 1, M. Caldin, Dr Med Vet, PhD, ECVCP Dip 2
1
Laboratorio d’Analisi Veterinarie San Marco, Padova, Italia
2
Clinica Veterinaria San Marco, Padova, Italia
Area di interesse: Leishmaniosi
Purpose of the work. Due to the limited effectiveness of most of the available methods for canine visceral leishmaniasis treatment and control, the
development of a dog vaccine is most desirable, practical and an ef?cient
control tool. This development reduces the dog-sand?y-dog peridomestic
transmission cycle and lowers the risk of transmission to humans. The protective potential of the excreted/secreted antigens of L. infantum promastigotes (LiESAP) was demonstrated in dogs (Lemesre et al., 2007). A
LiESAP/QA-21 vaccine has been recently authorized in the European
Union under the trade name CaniLeish (Virbac, France). It is composed of
purified excreted-secreted proteins of Leishmania infantum, produced by
means of a patented cell-free, serum-free culture system (Moreno et al.,
2012). According to European public assessment reports (EMA, 2011a;
EMA, 2011b), only purified excreted secreted antigens of Leishmania infantum promastigotes and an adjuvant should be present in the vaccine.
This purification is of crucial importance on the grounds that the secreted
and surface proteins that are exposed, can be processed by the host immune
system, unlike the intracellular proteins which are not. Furthermore, the release of internal proteins may function in conjugation with the secreted and
surface proteins acting as a transient “smoke screen” that enables the onset of the initial infection by viable parasites (Santarém et al., 2007). The
aim of the study was to investigate the presence of unexpected DNA from
Leishmania in vaccine vials.
Materials and used methods. Real-time PCR for L. infantum DNA detection
was performed in 7 vials belonging to two different lots and acquired through
standard commercial veterinary suppliers. We used primer and probe sequences specific for the L. infantum kinetoplast mini-circle (TIBMOLBiol,
Genua, Italy) as previously described (Solano-Gallego et al., 2007; SolanoGallego et al., 2012). Real-time PCR was performed by using LightCycler
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FastStart DNA Master, Hybridization Probes (Roche, Mannheim, Germany)
using LightCycler version 3.5.17 instrument (Roche, Mannheim, Germany).
Positive and negative controls were used in all runs. A conventional PCR
for ITS gene and direct sequencing were performed on each samples tested by real-time PCR (Schoenian et al., 2003). Sequences were compared
with sequences deposited in GenBank using BLAST hosted by the National
Center for Biotechnology Information, National Institutes of Health, USA
(http://www.ncbi.nlm.nih.gov).
Outcomes. High concentrations of Leishmania infantum DNA were found
during real-time qPCR based on kinetoplast multilocus gene on all samples
(7/7). Direct sequencing from conventional PCR for ITS gene revealed the
presence of Leishmania infantum with 100% homology with sequences deposited in GenBank database (FN398341).
Conclusions. Although, to our knowledge, very limited data is available in
the current veterinary literature related to the only commercially available
vaccine against canine leishmaniasis, the official documentation clearly
states that the product is highly purified and protein-free other than LiESAp
(EMA, 2011). Utilizing a protocol validated for samples other than canine
whole blood, enabled us to obtain significant amounts of Leishmania infantum DNA in all samples. Upon confirmation by others, our finding could
raise some concern about the efficacy of the purification process of the
product. If at all the presence of Leishmania DNA in the vial is considered
to be an expected finding, this would greatly emphasize the necessity to provide every detail of pertinent information to the scientific community before
or concurrently with the marketing of any innovative veterinary drug or
vaccine.
The presence of kDNA in the vaccine could cause further debate, because we
can not exclude, almost theoretically, a false positive PCR result in a sample
from a recently immunizated dog.
Bibliography
Lemesre JL et al., Long-lasting protection against canine visceral leishmaniasis using the
LiESAp-MDP vaccine in endemic areas of France: Double-blind randomized efficacy
field trial, Vaccine 25 (2007) 4223–4234.
European Medicines Agency [EMA] (2011a), CVMP summary of positive opinion for
Canileish, www.ema.europa.eu, accessed January 10th, 2013.
European Medicines Agency [EMA] (2011b), CaniLeish: EPAR – Product Information
(EMEA/V/C/002232), www.ema.europa.eu, accessed January 10th, 2013.
Moreno J et al. (2012), Use of a LiESP/QA-21 Vaccine (CaniLeish) Stimulates an Appropriate
Th1-Dominated Cell-Mediated Immune Response in Dogs, PLoS Negl Trop Dis 6(6):
e1683.
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Santarém N et al. (2007), Immune Response Regulation by Leishmania Secreted and Nonsecreted Antigens, Journal of Biomedicine and Biotechnology, Volume 2007, Article ID
85154, 10 pages.
Solano-Gallego L et al. (2007), Detection of Leishmania infantum DNA by fret-based real-time
PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol, 147(3–4):315
Solano-Gallego L et al. (2012), Detection of Leishmania infantum DNA mainly in Rhipicephalus sanguineus male ticks removed from dogs living in endemic areas of canine
leishmaniosis, Parasites & Vectors 2012, 5:98, 6 pages.
Schoenian G et al. (2003), PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003 Sep;47(1):349-58.
Dott. Tommaso Furlanello - Clinica Veterinaria San Marco, Via Sorio, 114/C,
35141 Padova (PD), Italia - Tel. ++390498561098 - E-mail: tf@sanmarcovet.it
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MISDIAGNOSI DI LINFOMA IN UN CANE
CON LEISHMANIOSI
F. Ibba, DVM1
1
Libero Professionista, Cagliari, Italia
Tipologia: Caso Clinico
Introduzione. Leishmania infantum è un protozoo trasmesso in Italia da vettori appartenenti al genere Phlebotomus spp., che causa nel cane una malattia
sistemica caratterizzata da una notevole varietà di sintomi clinici, specialmente cutanei. Questo case report dimostra come in corso di leishmaniosi sia
possibile osservare la comparsa, a livello cutaneo, di lesioni nodulari molto
insidiose, capaci di mimare molto da vicino un processo neoplastico linfoide,
causa di notevoli problemi diagnostici. Ciò appare importante soprattutto se
si considera che nel cane si sospetta una possibile correlazione fra infezione
da leishmania e insorgenza di linfomi.
Descrizione del caso. Dora è un cane femmina di 4 anni, di razza Bulldog
Inglese. Viene portato a visita per via di numerosi noduli cutanei di dimensione variabile da pochi mm a diversi cm di diametro. Alcuni di questi
noduli, localizzati in aree sottoposte a frizione, ulcerandosi hanno provocato abbondante sanguinamento. Dall’anamnesi risulta che le masse sono apparse in seguito a terapia con doxiciclina per rickettsiosi (rickettsia conorii),
instaurata un anno e mezzo prima presso diversa struttura. I reperti più significativi alla diagnosi erano anemia lieve normocitica normocromica, iperproteinemia, trombocitopenia non confermata da striscio.
Contestualmente il cane risultava sierologicamente negativo per ehrlichia
canis e leishmania (test rapidi). Dopo due settimane di terapia comparivano
delle masse distribuite sull’intera superficie corporea. Citologicamente
veniva classificato come neoplasia rotondocellulare maligna di probabile
origine linfoide o istiocitaria.
Sei mesi dopo sono stati eseguiti altri esami, in vista di una anestesia per
l’asportazione chirurgica di un nodulo da sottoporre a istologia. Venivano
ripetuti test sierologici per ehrlichia canis (IFAT < 1:32) e per leishmania
spp. (IFAT < 1:80). L’anemia persisteva, la trombocitopenia era scomparsa.
L’esame istologico della neoformazione non permetteva di distinguere con
certezza fra linfoma e neoplasia istiocitaria, ma l’immunoistochimica dimostrava che la quasi totalità della popolazione era composta di elementi T
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cd3 e cd 79 positivi. Nel corso dell’ultimo anno, le masse cutanee sono temporaneamente scomparse in due distinti periodi. Questo particolare, unitamente ad altri sintomi cutanei minori difficilmente attribuibili a linfoma, fa
dubitare della diagnosi originaria, nonostante la diagnosi istologica e istochimica. Un nuovo esame citologico delle lesioni permette ancora una
volta di identificare una popolazione di cellule di grandi dimensioni, con
citoplasma basofilo finemente vacuolizzato, nuclei rotondi o convoluti senza evidenti nucleoli, classificabili come elementi linfoidi immaturi. L’immunocitochimica dimostra nuovamente la loro appartenenza alla linea T. Si
effettuano inoltre ulteriori esami non eseguiti nelle occasioni precedenti,
quali l’elettroforesi delle proteine sieriche, che mostra una gammopatia
policlonale, e un esame del midollo osseo, che mostra un infiltrato linfoplasmocitario e permette di identificare amastigoti di leishmania. In attesa
di una prova più definitiva sull’origine neoplastica delle masse linfoidi cutanee (test di clonalità) viene eseguita la terapia leishmanicida. In breve
tempo le masse scompaiono completamente e i proprietari riferiscono di un
netto miglioramento della qualità di vita del cane.
L’esito della PCR per clonalità linfoide ha mostrato policlonalità dei linfociti B e oligoclonalità dei linfociti T.
L’assenza di monoclonalità genica permette di escludere una forma linfomatosa mentre l’oligoclonalità della linea cellulare T può essere attribuita
all’infezione da Leishmania/grave reattività linfocitaria. Il cane guarisce
perfettamente con la terapia antimoniale, scompaiono anche altri sintomi
minori (cheratite, dermatosi), e si assiste a un considerevole miglioramento
del quadro clinicopatologico. Dopo circa 15 mesi dall’inizio della terapia il
cane è ancora in remissione completa. L’esame citologico dei campioni
delle masse e dei linfonodi non ha permesso neppure a posteriori l’individuazione di amastigoti di leishmania. Non è stato possibile eseguire test di
biologia molecolare verso leishmania.
Conclusioni. Nonostante il quadro sintomatologico in corso di Leishmaniosi
canina appaia piuttosto vario, l’occorrenza di una forma nodulare cutanea non
è frequente. Quando ciò si verifica il quadro citologico appare più francamente istiocitario con abbondante componente linfoplasmocellulare ed è comune il reperimento di organismi riferibili a leishmania.
Questo caso evidenzia la possibilità di misdiagnosi, così come avviene in
medicina umana, in caso di neoformazioni linfoidi cutanee e sulla necessità di
ricorrere a strumenti diagnostici più complessi quali i test di biologia molecolare sulla clonalità linfocitaria per ottenere una diagnosi certa di linfoma
specialmente in aree endemiche di leishmania.
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Bibliografia
A Kawakami et al. Visceral leishmaniasis misdiagnosed as malignant lymphoma. InternMed
(1996) Volume: 35, Issue: 6, Pages: 502-506.
Böer et al. Unusual histopathological features of cutaneous leishmaniasis identified by polymerase chain reaction specific for Leishmania on paraffin-embedded skin biopsies. Br J
Dermatol 2006; 155:815-9.
Saab et al. Cutaneous leishmaniasis mimicking inflammatory and neoplastic processes. Journal of Cutaneous Pathology. Vol 39, Issue 2, pag 251–262, Feb. 2012.
Indirizzo per corrispondenza:
Dott. Fabrizio Ibba - Ambulatorio Veterinario Dr. Ibba Fabrizio Strada 40 N. 5,
Località Poggio Dei Pini, 09012 Capoterra (CA), ITALY
Tel. 070/725552 - Cell. 333 9116263 - E-mail: palmasoriano@hotmail.it
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EPIDEMIOLOGY, SEROPREVALENCE AND MANAGEMENT
OF CANINE VISCERAL LEISHMANIASIS IN PARAGUAY BY
THE NATIONAL PROGRAMME OF ZOONOSES CONTROL
J. Miret, DVM, MSc 1, L. Sosa, DVM 1, E. Galeano, DVM 1, H. Ocampos,
DVM 1, R. Martínez, DVM 1, J. Ojeda, DVM 1, M. Castagnino, DVM 1
1
Leishmaniasis Department. National Programme of Zoonoses Control and
National Rabies Center. Ministry of Public Health and Social Welfare,
San Lorenzo, Paraguay
Work type: Original Research
Topic: Leishmaniasis
Purpose of the work. Visceral leishmaniasis (VL), also called kala-azar, is an
infectious disease with chronic progression (Shaw, 2006). The causal agent of
VL is the protozoon Leishmania (Leishmania) infantum. In Brazil, as in other
places, VL transmission takes place during the heteroxenous life cycle of the
parasite. The major VL infection route for humans or animals is through the
bites of female phlebotomine sand flies, particularly of the species Lutzomyia
longipalpis (Lainson and Shaw, 1987). Dogs are considered to be the main
domestic reservoirs of the parasite, playing an important role in the epidemiological cycle of VL transmission to human hosts (Dantas-Torres, 2007). Canine leishmaniasis is an endemic disease, previous studies showed a high
prevalence of this diseases in Paraguay (Sosa et al., 2010; Miret et al., 2010).
The purpose of this work was to determine the seroprevalence of canine
Leishmaniasis by immunochromatographic rK39 test in serum samples obtained for routine exam requested by dog´s owners and veterinarians, active
surveillance in areas of silence transmissions and control of human cases notified for the National Service of Paludism Erradication (SENEPA) to the National Programme of Zoonoses Control and National Rabies Center (PNCZyCAN) of the Ministry of Public Health and Social Welfare (MSPyBS) in 2012.
Materials and used methods. A total of 12517 canine blood samples were analyzed by immunochromatographic rK39 test (Cypress®, Belgium), in the laboratory of Leishmaniasis of the PNCZyCAN coming from 11/17 departments
of the Paraguay. The statistical results were analyzed by Epiinfo 3.5.1.
Outcomes. The 5340 blood samples coming from routine exam from Asunción
(capital city) and the departments: Central, Boquerón, Caaguazú, Cordillera,
Itapúa, Guairá, Presidente Hayes, Paraguarí, Misiones, Alto Paraná and
Caazapá showed 2405 positive serum samples with a prevalence of (45.03%)
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of canine leishmaniasis. The active surveillance coming from Asunción and
the departments: Central and Misiones showed that 350 out of 2270 dog´s
blood samples were identified as positive with a prevalence of (15.41%) of canine leishmaniasis. From the 56 focus of human visceral Leishmaniasis cases, coming from Asunción, and the departments Central, Cordillera and
Paraguarí, it was observed that 791 out of 4907 dog´s blood samples showed
a prevalence of (16.11%) of canine leishmaniasis. It was observed a global
prevalence of (28.32%). Euthanasia procedures were performed in 273 positive dogs (78%) coming from active surveillance in areas of silence transmission and 492 positive dogs (62.19%) coming from focus of human visceral
leishmaniasis.
Conclusions. The high canine visceral Leishmaniasis shows the urgent need
to continue a strict epidemiological surveillance, sanitary education and
community participation by the Ministry of Public Health and Social Welfare
(MSPyBS) in the control of this disease in Paraguay.
Bibliography
Shaw JJ. Further thoughts on the use of the name Leishmania (Leishmania) infantum chagasi
for the aetiological agent of American visceral leishmaniasis. Mem Inst Oswaldo Cruz
2006;101(5):577-579.
Laison R, Shaw JJ. Ecology and epidemiology: New World. In: Peters W, Killick-Kendrick R,
editors. The Leishmaniasis in Biology and Medicine. London: Academic Press Inc; 1987.
p. 291-363.
Dantas-Torres F. The role of dogs as reservoirs of Leishmania parasites, with emphasis on
Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Vet Parasitol
2007;149(3-4):139-146.
Sosa L, Castagnino M, Miret J. Ocurrence of canine leishmaniasis and risk factors associated
in the city of San Lorenzo after the interventions of focus of cases of human visceral
leishmaniasis (year 2009). Rev Par Epidemiol. 2010; 1(S1): 74.
Miret J, Sosa L, Galeano E, Ocampos H, Martínez R, Ojeda J, Castagnino M. Epidemiological
situation of canine leishmaniasis in Paraguay (years 2005-2010). Rev Par Epidemiol.
2010; 1(S1): 74-75.
Dr. Jorge Miret - Programa Nacional de Control de Zoonosis y Centro Antirrábico Nacional.
Ministerio de Salud Pública y Bienestar Social, Ruta Mariscal Estigarribia Km 10,5
Campus UNA., San Lorenzo, Paraguay - Phone 59521422334 - Mobile 595971731242
E-mail: jorgemiret@gmail.com
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RAPID STIMULATORY EFFECT OF LEISGUARD®
ON THE CANINE INNATE IMMUNE SYSTEM
D. Sabaté, DVM, MSc, PhD 1, I. Mayós, PhD 1,
E. Cepeda, DVM 2, H. Josep, DVM, MSc, PhD 1
1
ESTEVE veterinaria R&D (Lab. Dr. ESTEVE, S.A.), Barcelona, Spain
2
Dep. Biologia Celular i Fisiologia (Universitat Autònoma de Barcelona),
Barcelona, Spain
Purpose of the work. Leisguard® is a domperidone based oral suspension
whose preventive efficacy against canine Leishmaniosis (CanL) has been
demonstrated in several studies (Llinás J et al. 2011, Gómez-Ochoa et al.
2012a, Sabaté et al. 2012). Its repeated administration to healthy dogs during 30 consecutive days results in a stimulatory effect on the innate immune
system, first defense barrier against the infection, being this the rationale for
its clinical efficacy. More precisely, its active principle, domperidone, increases the percentage of activated phagocytic cells for at least one moth after treatment conclusion (Gómez-Ochoa et al. 2004, 2012b) leading to a significant increase of their anti-Leishmania activity in case of contact with the
parasite (Gómez-Ochoa et al. 2013).
Given that the above mentioned stimulatory effect is evidenced early after
treatment initiation (Gómez-Ochoa et al. 2004), Leisguard® has been claimed
to be a practical alternative to vaccination against CanL for preventing dogs
living in non-endemic geographical areas occasionally travelling to endemic
areas for short periods of time.
The objective of this study was to accurately determine how fast is the stimulatory effect of Leisguard® on the dog’s innate immune system and for how
long it remains active in healthy animals. This information in essential to establish an adequate prophylactic approach against CanL for travelling dogs.
Materials and used methods. A total of 10 mixed breed healthy dogs of different sex (5 male and 5 female), age (3-12 years old), weight (8-31kg bw)
were included in the study with the consent of their owners. All dogs were
seronegative for anti-Leishmania antibodies (confirmed by a quantitative
ELISA test both at the beginning and at the end of the study).
The study had a duration of three months. During the first month (day 0 to 30)
all dogs were orally administered 1ml/10kg/day of Leisguard® (equivalent to
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0.5mg domperidone/kg/day). Blood samples were obtained at days 0, 1, 2, 3,
4, 5, 15, 30, 42, 58 and 92 in lithium heparin tubes and processed for the Nitroblue Tetrazolium test (NBT) following the instructions of the manufacturer
(Sigma). The NBT is an assay based on the activation percentage of neutrophils in peripheral blood that has been proposed for the follow up of canine leishmaniosis owing to the narrow relationship between the molecules
involved in the oxidative burst and the leishmanicidal activity of phagocytes
(Gómez-Ochoa et al. 2010, Scarpona et al. 2010).
Mean percentage of activated neutrophils (NBT positive) at each day was
compared to that of day 0 using the Repeated Measures ANOVA and the posthoc Holm-Sidak method with an overall significance level of 0.05.
Outcomes. A low baseline percentage of activated neutrophils was detected
in all dogs, prior to the first administration of Leisguard®, being this observation consistent with the results of previously published studies in healthy
dogs (Gómez-Ochoa 2004, 2012b). From the first day of Leisguard® administration and during the 30-day administration period a progressive increase in
the mean percentage of activated neutrophils was detected, with the highest
percentages of activation being recorded on days 15 and 30. After treatment
conclusion, mean percentage of activated neutrophils remained high up to
day 58 and then decreased towards the baseline levels, which were reached
on day 92. These results were again consistent with that reported in the literature for healthy dogs.
The statistical analysis of the results evidenced significant differences in
mean percentages of activated neutrophils vs. day 0 on day 2 (p=0.03), day 3
(p=0.006), days 4, 5, 15 and 31 (p<0.001), day 42 (p=0.011) and day 58
(p=0.014), therefore evidencing an extremely early stimulatory effect of Leisguard® on the dog’s innate immune system that persists at least two month after treatment initiation.
Conclusions. Administration of Leisguard® to healthy dogs results in a statistically significant stimulatory effect of the dog’s first defense barrier
against Canine Leishmaniasis from the second day of treatment onwards.
Consequently, dogs travelling to endemic areas can start treatment with Leisguard® very shortly before the trip or even upon arrival at their destination.
Bibliography
Gómez-Ochoa, P.; Gascón, M.; Castillo, J.A. (2004) Doctoral Thesis. Universidad de Zaragoza.
Gomez-Ochoa, P.; Lara, A.; Couto, G.; Marcen, J.M.; Peris, A.; Gascon, M.; Castillo, J.A.
(2010) Vet. Parasitol. 172, 135–138.
Gómez-ochoa, P.; Sabaté, D.; Homedes, J.; Ferrer, L. (2012a) Proceedings of the 73º Congresso Internazionale Multisala SCIVAC, Rimini, p. 545.
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Gómez-Ochoa, P.; Sabaté, D.; Homedes, J.; Ferrer, L. (2012b) Veterinary Immunology and Immunopathology 146:97-99
Gómez-Ochoa et al.; Sabate, D.; Homedes, J. (2013) Proceedings of the International Congress
on Canine Leishmaniasis and Vector-Borne Diseases SCIVAC, Pisa
Llinás, J.; Gómez-Ochoa, P.; Sabaté D.; Homedes, J. Ferrer, L. (2011) Proceedings of the
SEVC-46th AVEPA Congress, Barcelona.
Sabaté, D.; Llinás, J.; Suay, P.; Homedes, J.; Ferrer, L. (2012) Proceedings of the SEVC-47th
AVEPA Congress, Barcelona.
Scarpona, S., Romei, F., Di Cicco, E., Rossi, G. (2010) Proceedings of the 2nd International
Congress on Canine Leishmaniasis SCIVAC, Pisa, p. 167-168.
Dr. David Sabaté - ESTEVE Veterinaria R&D - Clinical Development, Pg.
Maragall, 154, 08027 Barcelona, Spain - Phone +34629358176 - E-mail: dsabate@esteve.es
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SEROLOGICAL AND CLINICAL EVIDENCE
OF LEISHMANIOSIS IN A DOG POPULATION LIVING
IN A FARM IN NORTHERN SERBIA
S. Savic, Phd, DVM 1, B. Vidic, Phd, DVM 1, Z. Grgic, Phd, DVM 1,
B. Gioia, PhD 2, L. Gradoni, Professor 2
1
Scientific veterinary institute “Novi Sad”, Novi Sad, Serbia
2
Istituto Superiore di Sanità, Rome, Italy
Purpose of the work. Scope of work: Determination of possible leishmaniosis in dogs which are in cohabitation with dogs diagnosed as positive, in the
northern Serbia region.
Introduction: Zoonotic visceral leishmaniosis is a vector borne disease mostly considered as a “Mediterranean disease”1,5. Serbia is considered a country with no autochthonous cases of leishmaniosis, only imported cases are
recorded in dogs and rarely in humans, mostly acquired in Greece and Montenegro during summer holidays. Phlebotomine vectors have been detected in
the southern part of Serbia many years ago6, and in the northern part there is
no evidence of their presence so far. Recently, the presence of vectors has
been identified in Hungary, close to the border with Serbia2. Also some serologicaly positive cases were found in bordering countries such as Bulgaria
and Croatia4,5.
Diagnostics of leishmaniasis is not always easy, it relys on clinical manifestation of the disease and serological findings3.
Materials and used methods. Materials and method: A small study was done
on 18 dogs living in the northern part of Serbia. The first group consisted of
16 dogs. They were of different ages (1 to 15 years old), sexes (5 males) and
breeds. Three of the dogs were imported from Greece, more then 10 years before (11, 14 and 15 years old) and since then, they have never left the farm.
Dog number 18 was an exhibition dog and he was taken for dog shows
abroad. Bitch number 17 lived with dog 18 and has never left the surrounding where she lived.
Blood samples were taken from dogs, to be tested for leishmaniosis by serology (ELISA and IFAT). ELISA was done with the commercial kit Euro Clone
Leishmania donovani IgG canine Elisa kit. IFAT was performed by an inhouse assay to re-test samples.
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Outcomes. Results: ELISA test results were interpreted as negative, weak positive or strongly positive, while IFAT results were interpreted at cut-off titre of
1/40-1/80 for “exposure” or “early infection“, and of =1/160 for “infection”.
After interpretation, the following was gained with ELISA test from the group
of 16 dogs:
- 3 dogs that were imported from Greece were positive
- 4 more dogs from that group had specific antibodies against leishmaniosis
- 1 was weak positive
- 8 dogs were found negative.
All of the ”newly” positive dogs were 3 or 4 years old. One of them was from
an offspring of the bitch imported from Greece, detected as positive.
In the case of dogs labelled as 17 and 18, both of them were strongly positive
with ELISA test.
After the interpretation of IFAT results from the group of 16 dogs:
- 4 dogs were found as “exposed” or “early infected” (1/40-1/80) (suspicious
or weak positive by ELISA)
- 2 dogs were found negative (suspicious by ELISA)
- 1 dog was found negative (negative by ELISA)
In the case of dogs 17 and 18, both of them were strongly positive by IFAT
(1/1280 and 1/2560).
Clinical signs were found in three dogs that came from Greece with positive
serological findings. One of them had elongated nails, skin lesions near labia
and cachexia. The other two dogs had skin lesions on the back and abdomen
and a very poor hair conditions. None of the dogs were ever treated and one
that came from Greece died recently.
Conclusions. Seven of 16 dogs (43%) were found positive, weak positive or
suspicious to leishmaniosis lived in the same farm and 4 of them have never
left home. Two of the dogs living in cohabitation were strongly positive to
leishmaniosis and one of them has never left home. There is evidence of clinical symptoms and serological findings of canine leishmaniosis in Serbia. In
4 cases of disease, the dogs from the study have been abroad at some point of
their life in countries where the disease is endemic (Greece, Italy and Montenegro).
In total 5 dogs were found with positive serological findings with ELISA and
IFA test for specific antibodies against Leishmania, that have never left their
homes and one of them even had clinical symptoms that could refer to leishmaniosis.
It is concluded that imported leishmaniosis exists in Serbia, but there are also cases of disease in dogs that did not leave the country.
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Bibliography
1. Bordoski A, Savin Z, Contribution to the study of the incidence of visceral leishmaniasis
in dogs in Montenegro, Acta parasitologica Jugoslavica 1, 1970, pg 33-36.
2. Farkas R, B.Tancoz, G. Bongiorno, M.Maroli, J.Dereure, P.D.Ready, First surveys to investigate the presence of canine leishmaniasis ind it’s Phlebotominae vectors in Hungary,
Vector-borne and zoonotic diseases Vol 11, No1, 2011, pg 1-12.
3. Paltrinieri S, L.Solano-Gallego, A.Fondati, G.Lubas, L.Gradoni, M.Castagnaro, A.Crotti,
M.Maroli, G.Oliva, X.Roura., A.Zatelli, E.Zini, Guidelines for diagnosis and clinical
classification of leishmaniasis in dogs, JAVMA Vol 236, No11, 2010, pg 1184-1191.
4. Tsachev I, E.I Papadogiannakis, V.Kontos, A.Ivanov, B.Chakarova, K.Strojanchev, R.Peshev, Seroepidemiology of Leishmania among healthy dogs in Bulgaria, Turkish Journal
of Animal Veterinary Science, 31(1), 2007, pg 73-74.
5. Zivicnjak T, F.Martinkovic, C.Khoury, G.Bongiorno, S.Bosnic, D.Lukacevic, M.Maroli,
Serological and entomological studies of canine leishmaniasis in Croatia, Veterinarski
arhiv 81 (1), 2011, pg 99-110.
6. Zivkovic V, Z.R.Adamovic, Phlebotominae (Diptera, Psyhodidae) of some natural microhabitats in Serbia, Acta Parasitologica Jugoslavica Vol 4 4/1973, pg 71-78.
Dr. Sara Savic - Scientific Veterinary Institute Department of Serology And Immunology,
Rumenacki Put 20, 21000, Serbia - Phone +381214895330 - Mobile +381638488469
E-mail: sara@niv.ns.ac.rs
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MYCOPLASMA HAEMOFELIS AND ‘CANDIDATUS
MYCOPLASMA HAEMOMINUTUM’ DISTRIBUTION
IN DISTINCT FELINE POPULATION, PORTUGAL
A. Duarte, PhD 1, V. Marques, Undergraduated student 2,
J. H. D. Correia, PhD 1, L. Tavares, PhD 1
1
Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade
de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal
2
Undergraduated student of Integrated Master Degree in Veterinary
Medicine, Faculdade de Medicina Veterinária,
Universidade de Lisboa, Lisboa, Portugal
Purpose of the work. Feline haemotropic mycoplasmosis is a bacterial disease of cats that can be found in a variety of other mammalian species. Three
major mycoplasma species have been described in cats, namely, Mycoplasma
haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’, being the first one the most pathogenic. Clinical signs
may include pallor, lethargy, anorexia, weight loss, depression and moderate
to severe anemia1. Several studies have been published about the prevalence
and epidemiology of these bacteria and nowadays it is believed to have a
worldwide distribution2. To our best knowledge only Mycoplasma haemofelis
has been reported in cats from Portugal and since only a few works have been
published in this country, more studies are needed.
The main goal of this study was to determine the frequency of haemoplasma
infections in cats from Portugal and to evaluate the correlations between infection and the geographic location, confinement/housing environment, age,
gender and FIV and/or FELV infections status.
Materials and used methods. Blood samples were collected from domestic and
feral cats, of both genders and originating from several confinement/housing environments, namely: domestic stray cats, feral cats (project CATUS), cats from
public shelters and from the Teaching Veterinary Hospital of the Veterinary Medicine Faculty (FMV) of the Technical University of Lisbon.
Blood was stored in EDTA immediately after collection, and DNA extraction
was carried out with the commercial kit DNeasy® Blood & Tissue Kit (Qiagen, Germany). After extraction, Mycoplasma haemofelis and ‘Candidatus
Mycoplasma haemominutum’ DNA was detected by qPCR using Taqman®
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probes3. The assay specificity for amplification of the two Mycoplasma species
was confirmed by direct amplicon sequencing.
Whanever possible, the presence of FIV antibodies and/or FeLV antigens
were tested by indirect ELISA (Virachek TM, Synbiotics).
Outcomes. A total of 260 cats were included in this study (95 cats from shelters,
21 stray cats, 34 feral cats and 110 domestic cats presented at the Veterinary
Teaching Hospital of FMV-UTL) of both genders and ages varying from 1-20
years old. When considering the total number of cats included, 14.62% (N=38)
were positive for Mycoplasma haemofelis. Frequencies of Mycoplasma haemofelis positive cats varied depending on the housing environment. None of the 21
stray cats included tested positive for this bacteria, while 25.26% (N=24 of 95),
3.64% (N=4 of 110) and 27.7% (N=10 of 36) of shelters, domestic and feral
cats, respectively, tested positive. ‘Candidatus Mycoplasma haemominutum’ was
present in 17.31% (N=45) of all cats tested. To our knowledge this is the first report of the presence of this species in mainland Portugal. For ‘Candidatus Mycoplasma haemominutum’, the detected frequencies varied differently from what
was observed for Mycoplasma haemofelis. Within the stray cats group 9,52%
(N=2 of 21) were positive; for shelters, domestic and feral cats the frequencies
found were 16,84% (N=16 of 95), 17,27% (N=19 of 110), 22.22% (N=8 of 36),
respectively. The presence of co-infection with both mycoplasma was present in
5.77% (N=15) of cats, half of which were shelter animals.
Conclusions. This is the first report of ‘Candidatus Mycolplasma haemominutum’ and Mycoplasma haemofelis frequency in different cat populations in
mainland Portugal. The prevalence of the two bacteria species was higher
than 15%. A thorough epidemiological survey will be necessary to fully understand the biological meaning of our findings.
Bibliography
1. Tasker, S., 2010, Haemotropic mycoplasmas what’s their real significance in cats. Journal of Feline Medicine and Surgery. 12, 369-381.
2. Sykes, J., 2010, Feline Hemotropic Mycoplasmas. Veterinary Clinics of North America:
Small Animal Practice. 40(6), 1157-1170.
3. Tasker, S., Helps, C., Day, M., Gruffydd-Jones, T., & Harbour, D., 2003, Use of Real-Time PCR to detect and quantify Mycoplasma haemofelis and “Candidatus Mycoplasma
haemominutum” DNA. Journal of Clinical Microbiology. 41(1), 439-441.
Corresponding Address: Prof.Ssa Ana Duarte - Faculdade De Medicina Veterinaria,
Universidade Tecnica De Lisboa Animal Health, Polo Universitário Da Ajuda,
Av. Da Universidade Tecnica, 1300-477 Lisboa, Lisboa, 1300-477, Portugal
Phone +351213652800 - E-mail: anaduarte@fmv.utl.pt
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HAEMATOLOGICAL PROFILES IN CANINE MONOCYTIC
EHRLICHIOSIS: A RETROSPECTIVE STUDY
OF 31 SPONTANEOUS CASES IN GREECE
I. Tsachev, DSc 1, D. Gundaheva, PhD 2, V. Kontos, PhD 3,
E. Papadogiannakis, PhD 4, S. Denev, DSc 5
1
Department of Microbiology, Infectious and Parasitic Diseases, Faculty
of Veterinary Medicine, Trakia University, Stara Zagora, BULGARIA
2
Department of Pathology, Faculty of Veterinary Medicine,
Trakia University, Stara Zagora, BULGARIA
3
Department of Veterinary Public Health,
National School of Public Health, Athens, GREECE
4
Department of Veterinary Public Health,
National School of Public Health, Athens, GREECE
5
4Department of Microbiology, Faculty of Agriculture,
Trakia University, Stara Zagora, BULGARIA
Work type: Clinical Case
Introduction. Canine monocytic ehrlichiosis is caused by the rickettsia
Ehrlichia canis. The disease is transmitted by the brown dog tick Rhipicephalus sanguineus and presents worldwide distribution [2, 9, 15].
Acute, latent and chronic forms of the disease are recognized, with the latter
usually accompanied by severe haematological and bone marrow dysfunctions [12].
Changes in the immune response to Ehrlichia canis are observed in all forms
of infection. In the acute form (approximately 2 months post-infection), serum
antibody titer can commonly reach high levels [10, 19]. The clinical picture
and haematological parameters have been reported in experimentally [7] and
naturally infected dogs [11], as well as in mixed (e.g ehrlichiosis and
babesiosis or ehrlichiosis and leishmaniosis) infections [14]. Thrombocytopenia, anaemia and leukopenia are usually the most common haematological abnormalities in canine monocytic ehrlichiosis. However, these laboratory changes are not specific for ehrlichiosis and may be present in other infectious diseases (e.g. leishmaniosis, bartonellosis etc).
The aim of the present study was to present the haematological parameters of
31 dogs with spontaneous monocytic ehrlichiosis in Greece and discuss the
results with the ones already known from the veterinary literature.
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Description of the case. The absolute number of platelets was found below
150x109/L in 35.48% of the samples examined, whereas in 58.07% of the cases these cells were found within the usual range (150-500x109/L). In a relatively low percentage of dogs (19.35%) the absolute number of erythrocytes
was found below 5.5x1012/L and HCT below 37%. Haemoglobin concentration was found below 120 g/L in 25.80% of dogs and the mean haemoglobin
content in erythrocytes (MCH) was depressed (< 19 pg) in 32.25% of dogs.
In addition, the mean haemoglobin concentration in erythrocytes (MCHC)
was low (< 320 g/L) in all examined samples and 90.32% of dogs have exhibited a large mean corpuscular volume (MCV > 77 fL). Hypochromia and
macrocytosis were also confirmed by microscopic analysis of blood smears in
93.54% and 90.32% of cases, respectively. Anisocytosis was present in
83.67% of cases. The erythrocyte distribution width was increased (>14.8%)
in the majority (80.64%) of samples tested.
The total leukocyte numeration was included in usual ranges in the majority
of diseased dogs (58.07%) but 35.48% of them exhibited leucopoenia (WBC
< 6.0x109 /L) and 6.45% exhibited leukocytosis (WBC > 17.0x109 /L). The
vast majority of animals (93.55%) presented no changes in the absolute number of lymphocytes and basophils, with lymphopenia and basophilia being detected in only 6.45% of animals. Neutropenia (< 3.6x109/L) was observed in
25.80% of samples. Monocytopenia (< 0.1x109/L) and eosinopenia (<
0.1x109/L) were found in 67.74% and 77.41% of samples, respectively.
Conclusions. Variations in haematological profiles in Ehrlichia canis infected dogs may be related to differences in the virulence of Ehrlichia canis
strains, antigen heterogeneity of this bacterial agent and the clinical form of
the disease. In addition, further studies are required to determine the influence of concomitant infections with other bacteria or parasites (e.g. Leishmania infantum, Bartonella sp., etc) on the observed haematological abnormalities.
Bibliography
GNONE A. ET AL.: Ehrlichiosis in anemic thrombocytopenic, or tick- infested dogs from a
hospital population in south Brazil. Vet. Parasitol., 2003, 117, 285-290;
FRANK J., BREITSCHWERDT E.: A retrospective study of Ehrlichiosis in 62 dogs from
North Carolina and Virginia. J. Vet. Int. Med., 1999, 13, 194-201;
HARRUS S. ET AL.: Presence of immune complex, and absence of antinuclear antibodies, in
sera of dogs naturally and experimentally infected with Ehrlihia canis. Vet. Microbiol.,
2001, 83, 343-349;
HARRUS S. ET AL.: Canine monocytes ehrlichiosis – an update. Comp. Contin. Educ. Pract.
Vet., 19, 1997, 431-444; HARRUS S. ?T AL.: Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. J. Clin. Microbiol., 1999, 37, 2745-2749;
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HARRUS S. ET AL.: Kinetics of the serum antiplatelet antibodies in experimental acute canine
ehrlichiosis. Vet. Immunol. Immunopathol., 1996, 51, 13-20;
HEERDEN V.: A retrospective study on 120 cases of canine ehrlihiosis. JSAVA, 1982, 53, 1722;
KUEHN N., GAUNT S.: Clinical and hematological finding in canine ehrlichiosis. JAVMA,
1985, 186 (4), 355-358;
MACIEIRA D. ET AL.: Prevalence of E. canis infection in thrombocytopenic dogs from Rio
de Janeiro, Brazil. Vet. Clin. Pathol., 2005, 34 (1), 44-48;
MURHY G. ET AL.: A molecular and serological survey of E.canis, E.chaffeensis and
E.ewingii in dogs and ticks from Oklahoma. Vet. Parasitol., 1998, 79, 325-339;
NEER T.: Canine monocytic and granulocytic ehrlihiosis. In: Infection disease of the dog and
cat, GREENE C. (ed), W.B.Saunders Co., Philadelphia, 1998, pp.: 139-147;
PIERCE K. ET AL: Acute canine ehrlihiosis: platelet survival and factor 3 assay. AJVR, 1977,
38, 1821-1825;
WANER T. ET AL.: Detection of plateled-bound antibodies in dogs after artificial infection
with Ehrlihia canis. Vet. Immunol. Immunopathol., 2000, 77 (1-2), 145-150;
WOODY B., HASKINS J.: Ehrlichial disease of dogs. VCNASAP., 1991, 21, 75-98.
Prof. Ilia Tsachev - Trakia University Epidemiology And Infectious Diseases, Bulgaria,
6000 Stara Zagora, Bulgaria - E-mail: ilia_tsachev@abv.bg
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ANAPLASMA PLATYS, EHRLICHIA CANIS
AND LEISHMANIA INFANTUM INFECTION
IN A SARDINIAN DOG INFESTED BY TICKS
A. Viglietti, DMV 1, C. Piazza, DMV 1, S. Tasca, DMV, ECVCP 2,
M. Caldin, DMV, ECVCP 2, M. Trotta, DMV, PhD 2
1
Libero Professionista, Carloforte (CI), Italia
2
Laboratorio Privato di Analisi Veterinarie “San Marco”, Padova, Italia
Tipologia: Caso Clinico
Introduction. In Sardinia (Italy) phlebotomine sandflies, mosquitoes, several ticks
species, lice and fleas and vector-borne diseases (VBDs), as canine Leishmaniasis and Ehrlichiosis, are common1,2,3,4. Co-infections by VBDs in clinical practice
are common, and have been described in serological or molecular report, but few
clinical descriptions are reported5. We describe a clinical case of co-infection by
Anaplasma platys, Ehrlichia canis and Leishmania infantum in a Sardinian dog
infested by Rhipicephalus sanguineus ticks.
Description of the case. A 4 year old male medium size crossbred dog from
Sardinia was evaluated for anorexia, severe weakness, vomiting, lethargy for
a few days and weight loss. On clinical examination the patient had tachypnea, slightly pale and dry mucous membranes, dehydration and hyperthermia. Several feeding ticks were found attached to the skin. Mild thrombocytopenia with platelets inclusions, suggestive with Anaplasma platys, was observed in blood smear; furthermore, acute renal failure, severe proteinuria
and dysproteinemia were found. PCR on blood sample resulted positive for
Anaplasma platys. Due to very severe clinical signs other vector-borne
pathogens were investigated by using serological and molecular analyses.
Co-infections by Ehrlichia canis and Leishmania infantum was revealed. Furthermore, serological positivity for Borrelia burgdorferi and Rickettsia
conorii was also found. Despite proper treatment the dog got worse and was
euthanized two days later. By the results of the autopsy and of the molecular
and serological test, a diagnosis of co-infection by Anaplasma platys,
Ehrlichia canis and Leishmania infantum was done.
Conclusions. During Anaplasma platys infection clinical signs are usually
mild and unspecific6, sometimes it is asymptomatic or it can causes fever,
lethargy, moderate limphoadenomegaly, weight loss, anorexia, depression,
pale mucous membranes7 and cyclic thrombocytopenia8.
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Several epidemiological studies have described co-infection with Leishmania
and Ehrlichia spp or Anaplasma spp: in these co-infections clinical signs and
laboratory abnormalities are usually more severe than single infection, with
high mortality6,9. In our case the dog showed slight abnormalities in CBC
count, but severe renal failure and inflammation too serious for Anaplasma
platys infection lonely: co-infection was suspected and concomitant monocytic ehrlichiosis and leishmaniasis were found, with severe clinical features as
reported in literature. It would be appropriate to consider Anaplasma platys
infection in dogs with anemia and or thrombocytopenia. Furthermore, in dogs
with severe symptoms6, compatible with vector borne diseases, consider also
co-infections: serological and molecular analyses can be useful tools for
helping clinician in revealing the specific agent causing disease.
Bibliography
1. Otranto, D., Dantas Torres, F., 2010. Canine and feline vector-borne disease in Italy: current situation and perpectives. Parasites and Vectors, 3:2.
2. Satta, G., Chisu, V., Cabras, P., Fois, F., Masala, G., 2011. Pathogens and symbionts in
ticks: a survey on thick species distribution and presence of thick-transmitted micro-organism in Sardinia, Italy. Journal of Medical Microbiology, 60, pp. 63-68.
3. Cocco, R., Sanna, G., Cillara, M.G., Tola, S., Ximenes, L.,Pinnaparpaglia, M.L., Masala,
G., 2003. Ehrlichiosis and Rickettsiosis in a canine population of Northern Sardinia. Ann
NY Acad Sci, 990, pp.126-130.
4. Solano-Gallego, L., Trotta, M., Razia, L., Furlanello, T., Caldin, M., 2006. Molecular survey of Ehrlichia canis and Anaplasma phagocytophilum from blood of dogs in Italy. Ann
NY Acad Sci, 1078, pp. 515-518.
5. Sasanelli, M., Paradies, P., Lubas, G., Otranto, D., de Caprariis, D., 2012. Atypical clinical presentation of coinfection with Ehrlichia, Babesia and Hepatozoon species in a dog.
Veterinary Record, vol 164, pp. 22-23.
6. Gaunt, S.D., Beall, M.J., Stillman, B.A., Lorentzen, L., Diniz, P.P.V.P., Chandrashekar,
R., Breitschwerdt, E.B., 2010. Experimental infection and co-infection of dogs with
Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings.
Parasites and Vectors, 3:33.
7. Harrus, S., Aroch, I., Lavy, E., Bark, H., 1997. Clinical manifestations of infectious canine cyclic thrombocytopenia. Veterinary Record, vol. 241, pp.247-250.
8. Harvey, J.W., Simpson, C.F., Gaskin, J.M., 1978. Cyclic thrombocytopenia induced by a Rickettsia-like agent in dogs. The Journal of infectious diseases, vol. 137, n°2, pp.182-188.
9. Koutinas, A.F., Mylonakis, M.E., 2010. Leishmaniosi canina complicate da malattie
trasmesse da zecche, comunemente presenti nei paesi mediterranei. In: Leishmaniosi canina: recenti acquisizioni su epidemiologia, implicazioni cliniche, diagnosi, terapia e prevenzione. Autori vari, EV (Eds.), Milano, pp. 75-80.
Corresponding Address: Dott. Antonio Viglietti - Ambulatorio Veterinario Associato
Dott. Viglietti-Dott.ssa Piazza, Via XX Settembre, 119, 09014 Carloforte (CI), Italia
Tel. 0781/857187 - Cell. 333/4651085 - E-mail: vigliettibus@hotmail.com
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POSTERS
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MOLECULAR SURVEY OF LEISHMANIA, EHRLICHIA,
ANAPLASMA AND BABESIA GENUS IN WOLVES
[CANIS LUPUS SIGNATUS] FROM NORTHEN PORTUGAL
G. M. Rosa, MSc 1,2, A. Duarte, PhD 3, C. Marques, MSc 3,
N. Santos, MSc 4, I. P. Fonseca, PhD 3, L. Tavares, PhD 3
1
Departamento de Biologia Animal, Faculdade de Ciências
da Universidade de Lisboa, Lisboa, Portugal
2
Department of Anthropology, Durrell Institute of Conservation
and Ecology, University of Kent, Canterbury, Kent,, United Kingdom
3
Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de
Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal
4
Life and Health Sciences Research Institute (ICVS) School of Health
Sciences, University of Minho (UMINHO), Braga, Portugal
Purpose of the work. Once distributed throughout the entire Iberian Peninsula, the Iberian wolf [Canis lupus signatus] population has been severely reduced.
Due to its warm climate, Portugal is an endemic country of several protozoa
and bacteria transmitted by arthropods such as ixodid ticks and phlebotomine
sand flies. In Northern Portugal, several of these pathogenic agents have already been described in domestic dogs, namely Babesia, Leishmania and
Ehrlicha/Anaplasma1.
Some of these protozoan and bacteria have significant veterinary medical importance and have already been described in wolves and foxes1-5, and may be
responsible for increased morbidity and mortality of endangered animal populations. On the other hand, wild animals may act as reservoirs contributing for
the sylvatic life cycle of these pathogenic agents.
In order to evaluate the distribution of these Vector Borne diseases we performed a molecular survey of parasites belonging to the Leishmania, Erlichia, Anaplasma, Ricketsia and Babesia genus in an opportunistic sample
collected from wild wolves in Northern Portugal.
Materials and used methods. Samples were collected between 1995 and 2011
by the Tissue Bank of Instituto de Conservação da Natureza (ICNb) Portugal,
from 43 wolves’ carcasses and included liver, spleen, bone marrow, skin and
lymph node and stored at -20ºC until processed.
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Total DNA was extracted using the commercial kit DNeasy ® Blood & Tissue Kit
(Qiagen, Germany). After extraction, detection of Leishmania spp., Anaplasma/
Ehrlichia spp., Babesia/Theileria spp. and Ricketsia spp. DNA was done
by qPCR using Taqman® probes.
Outcomes. Leishmania DNA was detected in fifteen visceral samples and only in one skin sample. Concerning Anaplasma/Ehrlichia spp. Thirty six positive samples were observed and six were positive for Babesia DNA. As for
Ricketsia only one sample tested positive.
Conclusions. To our knowledge this is the first report of vector borne diseases
in wild animals in mainland Portugal. Identification of positive samples
strengthens the potential role of these animals as disease reservoirs. A detailed serological and molecular survey should be performed in order to clarify the true epidemiological significance of these data.
Bibliography
1. Cardoso, L., Yisaschar-Mekuzas, Y., Rodrigues, F., Costa, A., Machado, J., Diz-Lopes, D.,
Baneth, G., 2010, Canine babesiosis in northern Portugal and molecular characterization
of vector-borne co-infections. Parasites Vect 3, 27.
2. Karbowiak G, Hapunik J, Miniuk M., 2008, The case of babesiosis in farmed wolf (Canis lupus L). Wiad Parazytol. 54, 243.
3. Leschnik M, Kirtz G, Virányi Z, Wille-Piazzai W, Duscher G., 2012, Acute granulocytic
anaplasmosis in a captive timber wolf (Canis lupus occidentalis). J Zoo Wildl Med. 43,
645-8.
4. Sastre N, Francino O, Ramírez O, Enseñat C, Sánchez A, Altet L., 2008, Detection of Leishmania infantum in captive wolves from Southwestern Europe. Vet Parasitol. 158, 117-20.
5. Dipineto, L., Manna, L., Baiano, A., Gala, M., Fioretti, A., Gravino, A.E., Menna, L.F.,
2007, Presence of Leishmania infantum in red foxes (Vulpes vulpes) in southern Italy.
Journal of Wildlife Diseases 43, 518-520.
Prof.ssa Ana Duarte - Faculdade De Medicina Veterinaria, Universidade Tecnica De Lisboa Animal Health, Polo Universitário Da Ajuda, Av. Da Universidade Tecnica,
1300-477 Lisboa, Lisboa, 1300-477, Portugal - Phone +351213652800
E-mail: anaduarte@fmv.utl.pt
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FLAVIVIRAL CENTRAL EUROPEAN TICK-BORNE
MENINGOENCEPHALITIS: NATURAL HISTORY AND
CLINICO-PATHOLOGICAL FEATURES OF 37 CASES
PRESENTED BETWEEN 1999 – 2012
L. Golini, Dr 1, C. Kleeb, Cand. Med. Vet. 1, F. Steffen, PD DECVN 1
1
Neurology Service, VetSuisse Faculty, University of Zurich, Zurich,
Switzerland
Purpose of the work. A Flavovirus (TBEV), transmitted by Ixodes ricinus,
causes the Central European Tick-Borne Encephalitis (CE-TBE). Forty-eight
consecutive cases have been diagnosed in our institution, between 1996 and
2012, detecting antibodies in the cerebrospinal fluid (CSF) directed against
TBEV antigen with a commercial ELISA (Klaus et al., 2011). Here we want
to describe the neurological clinico-pathological findings and outcome in
consecutive cases.
Materials and used methods. Thirty-seven complete medical records from
1999 and 2012 were available for review.
Outcomes. Thirty-four cases came from northeast Switzerland, three from
southwest Germany; referred between March and September and. The study
population was composed of several breeds from 1 to 13 years of age. All dogs
had an acute onset with, among others, fever (57%), neck pain (51%), hyperesthesia (16%), balance problems (46%), behavioral changes (32%), depressed mental status (27%), cranial nerves palsy (27%), and seizure activity (22%). Fever was associated with both presence of neck pain (p=0.37) and
multiple cranial nerve involvement (p=0.008). CSF showed pleocytosis (956.666 cells/µl, reference range <4 cells/µl) with variable protein content (0144 mg/dL; RR <35 mg/dL), and positive titer (8 – 175 UI/L; negative: < 5
UI/L). Cases were treated with steroids (12/37) or steroids and antibiotics
(16/37). Dogs without cranial nerves abnormalities (p=0.07) or balance
problems (p=0.04) were more likely to recover. On 27 available follow–up,
seventeen cases recovered after discharged and nine of those (52%) without
permanent sequelae. Abnormal mental status is associated with permanent
sequelae (p=0.01). Five died during hospitalization, five were euthanized after discharge.
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Conclusions. CE-TBE has to be considered in dogs with signs of encephalitis returning from endemic areas. Unlike previous reports (Tipold et al., 1993;
Leschnik et al., 2002), recovery can be expected however long-term sequelae
might last.
Bibliography
Tipold A., Fatzer R. and Holzmann H. Zentraleuropäische Zecken-enzephalitis beim Hund.
Kleintierpraxis 38:619-628, 1993.
Leschnik M.W., Kirtz G.C. and Thalhammer J.G. Tick-borne encephalitis (TBE) in dogs. Int.
J. Med. Microbiol. 291 (Suppl. 33): 66-69, 2002.
Klaus C., Beer M., Saier R., Schubert H., Bischoff S. and Süss J. Evaluation of serological tests
for detecting tick-borne encephalitis virus (TBEV) antibodies in animals. Berl Münch
Tierräztl Wochenschr 124, 444–449 (2011).
Dott. Lorenzo Golini - Vetsuisse Faculty, University Of Zurich Small Animal Surgery
Department, Neurology Service, Winterthurerstrasse 260, 8050 Zurich, Switzerland
E-mail: lgolini@vetclinics.uzh.ch
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HAEMATOLOGICAL CHANGES IN DOGS DURING THE
APPROPRIATE SEASON OF THE YEAR AS AN INDICATOR
FOR ACUTE CANINE BABESIOSIS
G. Kirtz, DVM 1, M. Leschnik, DVM 2, E. Hooijberg, DVM 1,
E. Leidinger, DVM, DipECVCP 1
1
INVITRO; laboratory for veterinary diagnostics and hygiene,
Vienna, Austria
2
Clinical Department of Small Animals and Horses,
Clinic for Internal Medicine and Infectious Diseases,
Veterinary University of Vienna, Vienna, Austria
Purpose of the work. Canine babesiosis is a protozoal infectious disease of
world wide importance. The aim of the present study was to evaluate haematological changes in canine blood in the acute phase of babesiosis and to confirm the biannual occurrence of Babesia canis, influenced by typical local climate conditions
Materials and used methods. A complete blood count from 358 dogs suspected of having babesiosis were evaluated (CELL-DYN. 3500 with Veterinary Package Software, Abbott Laboratories, IL, USA). An absolute reticulocyte count was performed in 154 dogs by microscopic evaluation as mentioned by Tvedten (21). A microscopic 100-cell differential was performed on
stained blood smears (Hemacolor®, Merck, Vienna, Austria). The presence of
Babesia canis was confirmed by microscopic examination of stained blood
smear by the concentration line technique. For statistical analysis (SPSS v.
14; SPSS Inc., Chicago, US) a classification and regression tree model
(CART) was calculated. For each case of babesiosis, climate data (mean daily air temperature, relative humidity, precipitation) recorded during the most
probable incubation time were analysed. Climate data were measured at a
meteorological station located near Vienna
Outcomes. Thrombocytopenia was present in all 113 dogs, with 55.8% of
these showing a severe thrombocytopenia. Red blood cell count, packed cell
volume and haemoglobin values were below the reference range in 62.8%,
61.1% and 46.0% of affected dogs, respectively. Slight regeneration was obvious in only 5 Babesia canis positive dogs. Leukopenia, lymphopenia, neutropenia and monocytosis were present in 54.9%, 47.8%, 30.4% and 6.5% of
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the dogs, respectively. Evaluating haematological parameters by CARTanalysis revealed a predictive model (accuracy= 93.5%) for canine babesiosis, when using the leucocyte, thrombocyte, and reticulocyte count. Climate
conditions resulted in a biseasonal accentuated occurrence of canine
babesiosis with preferential mean temperature development ranging from
8.8°C to 13.7°C in spring and 18.3°C to 8.1°C in autumn.
Conclusions. The results demonstrate that on the basis of typical haematological changes, at the appropriate time of the year, a tentative diagnosis of
canine babesiosis is achievable and further specific investigations should
follow.
Bibliography
Furlanello T, Fioro F, Caldin M, Lubas G, Solano-Gallego L. Clinicalpathological findings in
naturally occurring cases of babesiosis caused by large Babesia from dogs in northeastern
Italy. Veterinary Parasitology 2005; 134: 77-85.
Guelfi JF, Candebat D, Fourcade E, Thevenet D, Espie C. Variations de lhémogramme en function de lancienneté des symptoms chez les chiens adultes atteints de babésiose aiguë
spontanée. Revue de Médecine Vétérinaire 1998; 149 : 65-68.
Leschnik M, Kirtz G, Tichy A, Leidinger E.. Seasonal occurrence of canine babesiosis is influenced by locate climate conditions. International Journal of Medical Microbiology
2008; 298: 44 (Suppl), 243-248.
Leschnik M, Kirtz G. „Endemic“ Tick-borne diseases: clinical and laboratory aspects. In: Proceedings of the international congress: Small animal infection diseases. Smolenice, Slovakia, March 21 to 22. 2009; 103-105.
Protopopoff N, Van Bortel W, Speybroeck N, Van Geertruyden JP, Baza D, D’Alessandro U,
Cossemanns M. Ranking malaria risk factors to guide malaria control efforts in African
highlands. 2009. Public Library of Science One 4, e8022
Szell Z, Sréter-Lancz Z, Márialigeti K, Sréter T. Temporal distribution of Ixodes ricinus, Dermacentor reticulatus and Haemophysalis concinna in Hungary, Veterinary Parasitolology
2006; 141: 377-379.
Zygner W, Goska O, Rapacka G, Jaros D, Wedrychowicz H. Hematogical changes during the
course of babesiosis caused by large Babesia in domestic dogs in Warsaw (Poland). Veterinary Parasitolology 2007; 145: 146-151.
Dr. Georges Kirtz - Invitro Hematology/Cytology, Rennweg 95, Vienna/Austria/1030, Austria
Phone +431799622914 - E-mail: georges.kirtz@invitro.at
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ANAPLASMOSIS IN DOGS IN AUSTRIA:
A SEROPREVALENCE STUDY
G. Kirtz, DVM 1, E. Leidinger, DVM, DipECVCP 1
1
INVITRO; laboratory for veterinary diagnostics and hygiene,
Vienna, Austria
Purpose of the work. The aim of this retrospective study (2001 –2009) was to
determine the prevalence A. phagocytophilum infection in austrian dogs.
Materials and used methods. We examined the existence and level of IgG-antibodies against A. phagocytophilum in blood samples by an indirect immunofluorescence assay (IFAT) using A. phagocytophilum as antigen. We examined blood
samples for antibodies against A. phagocytophilum in 2959 dogs.
Outcomes. Antibodies against A. phagocytophilum were found in 1625 blood
samples (54.9%) by IFAT. One hundred and seventy seven (177) dogs had an
titer (1:40) below the cut off (1:80), so that these dogs were considered as
seronegative. The majority of the dogs affected A. phagocytophilum lived in,
or near by, regions, known as ticks areas.
Conclusions. Serological tests are often used to diagnose tick –borne diseases. However, patients in the acute phase of the disease are frequently
seronegative. Therefore, serological tests always should be done on paired
serum samples collected during the acute phase and 14 to 20 days later.
Anaplasmosis is diagnosed with increasing frequency in Austrian dogs since
2000. Tick-borne diseases normally occur in restricted areas, where infected
ticks live. In the last years an enlargement of the endemic areas where Ixodes
ricinus lives has been observed, this may be a possible reason that, in recent
years, A. phagocytophilum infection has increased in incidence and clinical
importance. Pets may represent a good biological indicator for the presence
and distribution of tick-borne diseases.
Bibliography
JENSEN J, SIMON D, MURUA ESCOBAR H, SOLLER JT, BULLERDICK J, BEELITZ P,
PFISTER E, NOLTE I (2005) Prevalence of anaplasma phagocytophilum in dogs in Germany. Proceedings of 15th ECVIM-CA Congess, Glasgow, UK., 2005, 943.
KIRTZ G, MELI M, LEIDINGER E, LUDWIG P, THUM D, CZETTEL B, KÖLBL S, LUTZ
H (2005) Anaplasma phagocytophilum infection in a dog: identifying the causative agent
using PCR. JSAP, 46, 300-303.
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KOHN B, SILAGHI C, GALKE D, ARNDT G, PFISTER K. Infections with Anaplasma phagocytophilum in dogs in Germany. Research in Veterinary Science. 2010, doi:10.1016/
j.rvse.2010.08.008.
PFISTER E, GALKE D, KOHN B (2006); Anaplasmose durch A. phagozytophilum- eine sich
ausbreitende Infektionskrankheit. Proceedings der 52. Jahrestagung der DGK-DVG, Düsseldorf, 21-24. Sept. 2006.
SOLANO-GALLEGO L, LLULL J, OSSO M, HEGARTY B, BREITSCHSCHWERDT E
(2006): A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet. Res.,37, 231-244
Dr. Georges Kirtz - Invitro Hematology/Cytology, Rennweg 95, Vienna/Austria/1030, Austria
Phone +431799622914 - E-mail georges.kirtz@invitro.at
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RETROSPECTIVE STUDY OF CANINE EHRLICHIOSIS
IN BUENOS AIRES
E. G. Maubecin, DVM 1, M. E. Pereira, DVM 1, M. M. Fidanza, DVM 1,
J. Carrizo, DVM 1, G. A. Mira, DVM 1, I. Lippi, DVM, PhD 2,
N. V. Gomez, DVM, PhD 1
1
School of Veterinary Sciences-University of Buenos Aires,
Buenos Aires, Argentina
2
Department of Veterinary Science - University of Pisa, Pisa, Italia
Purpose of the work. Canine Ehrlichiosis is an infectious disease, caused by
Ehrlichia canis. Ehrlichia are Gram-negative, pleomorphic, intracellular bacteria, with predilection for monocytes and polymorphonuclears. Ehrlichia organisms are transmitted through the bite of infected ticks, such as Riphicephalus
sanguineus (most common and widely distributed tick in dogs). Ehrlichiosis
represents a potentially fatal disease in dogs and other members of the Canidae
family, with non-specific clinical signs. The incubation period lasts for 9 to 14
days. The disease shows three phases: acute, subacute and chronic, which cannot be easily differentiated due to the similarity of clinical signs, such as pale
mucous membranes, inappetence, apathy, fever, lymphadenopathy,
splenomegaly. The finding of morulae of Ehrlichia Canis confirms the diagnosis of Ehrlichiosis, although their presence in blood is transitory, specially during acute phase1. For this reason serologic techniques represent the preferred
tools to detect the infectionThe aim of the present study was to report Ehrlichiosis in a new geographic location and to bring out the importance of the disease in the differential diagnosis of hematological alterations.
Materials and used methods. The present study was developed in 50 dogs of
different breed, age and gender presented in Buenos Aires between June 2011
and November 2012. The enrolled dogs showed hematological alterations,
hepato-splenomegaly and/or lymphadenopathies with or without a previous
history of ticks. Patients were submitted to complete clinical evaluation, CBC
and abdominal ultrasound and to bone marrow aspiration for the identification of morulae in the leukocytes through a Metanol–Giemsa stain. All dogs
were tested by a commercial ELISA test InmunoComb®, which detects IgG
against Ehrlichia Canis with a correlation of 94% with the indirect immunofluorescent test.
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Outcomes. Twenty-three dogs (46%) were positive to Ehrlichia Canis: 1/23
(4.34%) showed regenerative macrocytic, hypochromic anemia, while 18/23
(78.26%) normocytic, normochromic, non-regenerative anemia. In the second
group, 1/18 (5.55%) showed aplasia of red cells, 4/18 (22.22%) different degrees of hypoplasia of the three cellular lines and 6/18 (33.33%) pancytopenia
secondary to medullary aplasia. Leucocytosis with neutrophilia and left shift
was present in 6/23 (26%) dogs, while trombocytopenia in 9/23 (39.13%).
Morulae were detected in none of the patients, neither in peripheral blood nor
in bone marrow. Abdominal ultrasound detected splenomegaly in 9/23
(66.86%), hepatomegaly in 4/23 (17.39%) and abdominal lymphadenopathies
in 13.04%. In agreement with other reports2,3,4, peripheral blood cytopenia was
a common alteration in both forms of the disease. The differences between acute
and chronic Ehrlichiosis were evident at bone marrow aspiration. In acute form
regenerative anemia resulted from cellular destruction in peripheral blood and
from hypercellularity of bone marrow and only 5% presented morulae. Among
dogs in acute phase only one presented without morulae. The number of dogs
in chronic phase presenting non-regenerative anemia and leucopenia with different degrees of hipoplasia or aplasia of the bone marrow was higher than previously reported by other Authors3,4 .
Conclusions. The finding of canine Ehrlichiosis in Buenos Aires and its surrounding areas allows to become aware on the disease, which has not been
diagnosed previously in this zone, probably due to climatic changes and to environmental and socioeconomic factors.
Bibliography
1. Faria, JL; Dagnone, AS; Munhoz, TD; et.al. (2010): Ehrlichia canis morulae and DNA detection in whole blood and spleen aspiration samples. Rev Bras Parasitol Vet. 19:2, 98-102.
2. Hoyos L (2007): Evaluación del Examen Hematológico en el Diagnóstico de Ehrlichiosis canina. Rev. Inv. Vet. Perú. 18:2, 129-135.
3. Texeira, BM; Duarte, LS; Rabelo, GPN; Crissiuma, AL; Herdy, MA; Vasconcelos, TC
(2009): Frecuencia de mórulas de Ehrlichia Canis en frotis sanguíneo de 1394 perros (Canis familiaris) atendidos en el Hospital de la Escuela de Medicina Veterinaria de Unigranrio, Río de Janeiro, Brasil y demás alteraciones hematológicas encontradas. Memorias del Latin American Veterinary Conference, 2009. Lima, Perú, 43-437.
4. Lorente Méndez, C (2004): Tesis Doctoral: Evaluación hematológica e inmunofenotípica
de la “ehrlichiosis canina”: evolución tras la administración de “dipropionato de Imidocarb” Universidad Complutense de Madrid. Facultad de Veterinaria.
Corresponding Address: Dott.ssa Ilaria Lippi - Università di Pisa, Dipartimento di Scienze
Veterinarie, Via Livornese Lato monte, 56122 San Piero a Grado (PI), Italia
Tel. 050/2210100 - E-mail: ilaria.uk@hotmail.it
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CANINE LEISHMANIASIS: A COMPARATIVE STUDY OF
REAL-TIME PCR IN CONJUCTIVAL AND WHOLE BLOOD
SAMPLES AND INDIRECT FLUORESCENT ANTIBODY
TEST FOR THE DETECTION OF LEISHMANIA INFANTUM
A. Mountousi, BSc 1, M. Linou, DVM 1, V. Pogka, BSc, PhD 1,
P. Mpizta, BSc 1, M. Emmanouil, BSc, PhD 1,
A. Papaioannou, DVM, MSc 2, A. F. Mentis, MD, PhD 1
1
Hellenic Pasteur Institute, Athens, Greece
2
Papaioannou Veterinary Clinic, Markopoulo, Attiki, Greece
Purpose of the work. Canine leishmaniasis (CanL) caused by Leishmania infantum is an endemic zoonotic disease and dogs are considered to be the main
animal reservoir host. In many CanL cases, infection does not equal clinical
illness resulting in a high prevalence of subclinical infection1. Our aim was to
develop an in-house real-time PCR (rt-PCR) to use in addition to indirect fluorescent antibody test (IFAT) in order to detect L. infantum infection in dogs
living in a highly endemic area for leishmaniosis.
In treated dogs, IFAT is highly sensitive in detecting specific anti-Leishmania
antibodies, yet it does not provide information on latent infections, nor does
it have a good predictive value for relapses during the follow-up of treated
dogs. rt-PCR can be considered the most sensitive and simple assay for the
early detection of relapses and thus be used to monitor the evolution of infection after therapy2.
Materials and used methods. Conjunctival swabs (CS), whole blood and
serum samples from 73 dogs that were collected from the Diagnostic department of the Hellenic Pasteur Institute as well as from a collaborating veterinary clinic, were enrolled in this study.
IFAT was carried out using cultured promastigotes of L. infantum as antigen,
kindly provided by the Reference Center for Leishmaniasis (Hellenic Pasteur
Institute). Washed promastigotes were dispensed and fixed on multispot microscope slides (MD Biomedicals Inc.), air-dried and kept at -70°C until use.
Two-fold serial dilutions of canine sera were performed to determine the antibody titer.
Canine serum samples diluted in PBS (1:25) were added on the multispot
slides with L. infantum antigen and incubated in a moist chamber at 37 ± 2
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°C for 30 min. Fluorescein labeled purified rabbit anti-dog immunoglobulin
G (IgG) antibody (P.A.R.I.S, France) was added and the slides were incubated again in a moist chamber at 37 ± 2 °C for 30 min. Then, the slides were
washed with PBS and left to air-dry before microscopic observation. A cut-off
value =1:400 was used to establish a positive IFAT result.
CS and whole blood samples were analysed using a rt-PCR protocol targeting the kinetoplast DNA (kDNA) of the parasite3. Assays based on the detection of kDNA appear to be the most sensitive for direct detection in infected
tissues because this molecular target is present at about 104 copies per cell4,
5
. DNA was extracted using the commercial kits QIAamp DNA mini kit and
DNeasy Blood and Tissue according to manufacturer’s instructions (Qiagen,
Hilden, Germany).
Outcomes. Twenty one out of 73 (28.8%) dogs were positive for L. infantum
with IFAT and/or rt-PCR. More specifically, 7 dogs were seropositive and also presented with a positive rt-PCR result in both blood and CS samples, 1
dog had a positive IFAT and CS result but a negative result in whole blood rtPCR and 1 dog that was positive in CS rt-PCR but negative in IFAT and whole
blood rt-PCR. The remaining 12 dogs presented only with a positive IFAT.
Conclusions. Laboratory findings in addition to clinical manifestations could
discriminate those cases where a new infection is present from those dogs
with latent infections, thus avoiding unnecessary treatment. Furthermore, this
approach can be used to monitor the parasitological status of animals during
and after treatment. As frequent assessments are required for the above purpose, CS samples can be valuable for CanL diagnosis because they are both
sensitive and noninvasive6.
Even though IFAT is considered to be the gold standard method for CanL diagnosis, rt-PCR is regarded as a powerful tool for a more accurate laboratory evaluation. Further samples are needed to establish the importance of rtPCR to the interpretation of a clinical case leading to a better confrontation
of cases with uncertain outcomes.
Bibliography
1. Baneth G et al. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends Parasitol 2008; 24(7):324-330.
2. Solano-Gallego L. et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasites & Vectors 2011; 4:86.
3. Mary et al. Quantification of Leishmania infantum DNA by a real-time PCR assay with
high sensitivity. J Clin Microbiol 2004; 42(11):5249-55.
4. Manna L et al. Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. Vet J 2008; 177(2):279-282.
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5. Lambson B et al. Leishmania donovani: development and characterisation of a kinetoplast
DNA probe and its use in the detection of parasites. Exp. Parasitol. 2000; 94:15–22.
6. Di Muccio T et al. Diagnostic Value of Conjunctival Swab Sampling Associated with Nested PCR for Different Categories of Dogs Naturally Exposed to Leishmania infantum Infection. J. Clin. Microbiol. 2012; 50(8):2651.
Ms. Maria Linou - Hellenic Pasteur Institute Public Health, 127 Vassilisis Sofias Avenue,
Athens/Greece/11521, Greece - Phone 00302106478822 - E-mail: linoum@pasteur.gr
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LEISHMANIA INFANTUM PCR POSITIVE LYMPH NODE
ASPIRATES: CYTOLOGIC PATTERNS IN CATS
L. Perillo, DVM, PhD, MScs 1, M. G. Pennisi, DVM, professor 1,
L. Solano-Gallego, DVM, PhD, ECVCP 2, T. Lupo, DVM, PhD 3,
A. Migliazzo, DVM, PhD 3, G. Mazzullo, DVM, professor 1
1
Dipartimento di Scienze Veterinarie, Università di Messina, Messina, Italy
2
Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria,
Universitat Autonoma de Barcelona, Barcellona, Spain
3
C.Re.Na.L., IZS Sicilia “A. Mirri”, Palermo, Italy
Purpose of the work. Leishmaniosis caused by Leishmania infantum is an endemic zoonosis in the Mediterranean basin where domestic dogs are considered the major reservoir of the parasite.1 Cats living or travelling in this area
are considered an unusual host of the protozoan and a variable prevalence of
infection is reported in this species.2
Few studies report detailed descriptions of the lymph node cytological pattern
in canine L. infantum infection but the histopathological features are characterized by chronic lymphadenitis involving the entire architecture of the nodes
including the capsule, subcapsular sinus, cortical (lymphatic nodules and
paracortex) and medullary regions (medullary sinus and medullary cords).3
Data concerning the clinical-pathological findings in Leishmania infected
cats are scarce.
The aim of the present study was to describe cytological patterns of lymph
nodes in cats with clinical or subclinical forms of L. infantum infection diagnosed by real-time PCR.
Materials and used methods. Twenty cats (age range 7-132 months), with
(n=18) or without (n=2) clinical evidence of the disease at physical examination but with a L. infantum PCR positive lymph node aspirates were cytologically evaluated. CBC and serum protein electrophoresis were performed.
Lymph node fine needle aspirates were stained with May-Grümwald Giemsa
after air drying. Cytological evaluation was done scanning the entire slide at
10x and differential cell counts of 1000 cells were made at 40x. The presence
of amastigote forms of L. infantum was determined observing 300 fields at
100x magnification.
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munofluorescent antibody test (IFAT) and for FIV and FeLV infections (FASTest®
FeLV-FIV, Megacore Dagnostik, Austria).
Outcomes. Lymphoid hyperplasia was the only cytological pattern found in
all cats. Both intra and extracellular amastigotes of Leishmania were only observed in six seropositive cats with clinical leishmaniosis (IFAT titre range
160-2560). Eleven sick cats resulted IFAT positive for L. infantum (titre range
20-2560). All cats were FeLV negative and 4 cats were positive for FIV antibodies. CBC abnormalities were found in 9 cats out of 17 and consisted of
anemia (7 cats), leucopenia (1 cat), leucocytosis (1 cat) and thrombocytopenia (4 cats). Serum protein electrophoresis changes were evident in all cats:
hypoproteinemia (1 cat), hyperproteinemia (12 cats), hypoalbuminemia (4
cats), hyperalbuminemia (1 cat); globulin changes consisted in reduced a1 (3
cats), reduced a2 (1 cat), increased a2 (7 cats), reduced ß (9 cats), increased
ß (1 cat), reduced ? (2 cats), increased ? (3 cats) and reduced A/G (6 cats).
Conclusions. The observed cytological pattern consisting of lymphoid hyperplasia is similar to the one often reported in dogs with clinical leishmaniosis.4
Lymphoid hyperplasia was found in all investigated cats irrespective of their
clinical status and FIV co-infection. In addition, the sensitivity of PCR is
much higher than both serology and cytological evaluation as described in
dogs.5 Leishmania infection should be considered in the differential diagnoses
of lymphoid hyperplasia in cats living or travelling in endemic areas.
Bibliography
1. Baneth G., Solano-Gallego L., 2012. Canine leishmaniosis. In Greene CE ed. Infectious
diseases of the dog and cat. St. Louis: Saunders-Elsevier, 735-746.
2. Maroli M., Pennisi M.G., Muccio T., Khoury C., Gradoni,L., Gramiccia M., 2007. Infection of sandflies by a cat naturally infected with Leishmania infantum. Vet. Parasitol. 30,
357–360.
3. Lima W.G., Michalick M.S., de Melo M.N., Luiz Tafuri W., Luiz Tafuri W., 2004. Canine visceral leishmaniasis: a histopathological study of lymph nodes. Acta Trop.
92(1), 43-53.
4. Mylonakis M.E., Papaioannou N., Saridomichelakis M.N., Koutinas A.F., Billinis C.,
Kontos V.I., 2005. Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum. Vet. Clin. Pathol. 34(3), 243-7.
5. Moreira M.A., Luvizotto M.C., Garcia J.F., Corbett C.E., Laurenti M.D., 2007. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. Vet. Parasitol. 30,145(3-4), 245-52.
Corresponding Address: Dott.ssa Laura Perillo - Università di Messina, Via Andria 8,
98121 Messina (ME), Italia - Cell. 347/0877347 - E-mail: lauraperillo77@gmail.com
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STUDIO CLINICO RETROSPETTIVO SULLA PREVALENZA
SIEROLOGICA DELLE MALATTIE EMOPROTOZOARIE
IN UNA POPOLAZIONE CANINA
1
S. Pitarra, MV 1
Libero professionista, Fragagnano, Italia
Scopo del lavoro. Quantificare le malattie oggetto dello studio nel periodo
intercorso tra Gennaio 1996 e Dicembre 2006, in vista di una sempre maggiore diffusione legata agli attuali cambiamenti climatici. Sono patologie
considerate emergenti dal mondo scientifico, dal 1980 infatti la presenza dei
vettori, nel caso specifico delle zecche, si è estesa entro una maggiore latitudine e altitudine, gli agenti dei quali è stata riconosciuta la natura zoonosica
sono circa 200 (Acha e Szyfres, 1989). L’intento è stato quello di effettuare
un’istantanea del periodo analizzato con lo scopo di valutare la presenza di
queste malattie in vista di una futura maggiore diffusione, dato l’alto potenziale zoonosico.
Materiali e metodi. È stato eseguito uno studio retrospettivo relativo a un periodo compreso tra gennaio 1996 e dicembre 2006, su 2691 campioni di sangue canino, sottoposti al test dell’immunofluorescenza indiretta per Borrelia
burgdorferi, Ehrlichia canis, Rickettsia conorii e Rickettsia rickettsii. Per la
diagnosi di Hepatozoon canis è stato eseguito un esame citologico sullo striscio di sangue colorato con le tecniche di May Grunwald Giemsa® o di Diff
Quick®, identificando i gametociti nei neutrofili e nei monociti.
E’ stato utilizzato il siero per il test dell’immunofluorescenza indiretta (IFI).
Sono stati considerati positivi al test dell’immunofluorescenza indiretta i soggetti con titolo anticorpale superiore o uguale a:
• 1: 40 per Ehrlichia canis
• 1: 64 per Borrelia burgdorferi
• 1: 64 per Rickettsia conorii e per Rickettsia rickettsii
Sono stati presi in considerazione alcuni parametri di laboratorio comuni ai
casi positivi in possesso, in particolare: RBC, WBC, PLT, indici eritrocitari, Hct.
Per il profilo biochimico sono stati valutati i parametri di funzionalità epatica
e renale e le modificazioni delle proteine seriche attraverso: urea, creatinina,
ALP, ALT, AST, proteine totali.
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I soggetti costituenti il campione sono stati visitati presso un’unica struttura,
per loro è stata compilata una cartella clinica dettagliata e divisi per razza.
Risultati. L’analisi è stata eseguita su 2691 campioni di sangue di cui 410
(15%) sono positivi e 2281 (85%) negativi. Dei 410 test positivi, 237 (58%)
sono positivi a Ehrlichia canis, 106 (26%) a Rickettsia rickettsii, 27 (7%) a
Borrelia burgdorferi, 22 (5%) a Hepatozoon canis e infine 18 (4%) a Rickettsia conorii. A prima vista spicca che più della metà delle positività segnalate,
lo sono a Ehrlichia canis, che insieme a quelle relative a Rickettsia rickettsii
costituiscono l’84% (343/410) del campione. Questa netta preponderanza delle due malattie è dovuta al fatto che le positività sono state riscontrate per un
periodo di tempo più esteso, per Ehrlichia canis dal 1996 al 2006, mentre per
Rickettsia rickettsii dal 1999 al 2006. Le positività alle altre malattie risultano testate solo in alcuni anni, per Borrelia burgdorferi (1998-2003), per Hepatozoon canis (1996-1997), per Rickettsia conorii (2003-2006). Inoltre, per
ogni malattia sono stati eseguiti un numero di test diversi, in particolare:
o Ehrlichia canis 52% (1399/2691)
o Rickettsia rickettsii 27% (729/2691)
o Borrelia burgdorferi 11% (288/2691)
o Hepatozoon canis 6% (146/2691)
o Rickettsia conorii 4% (129/2691).
Per avere una panoramica generale si sono sommati i test positivi a tutte le
malattie per ogni anno esaminato, ne è risultato che dal 1996 al 1999 le positività hanno avuto un andamento costante con circa 20 casi positivi, per avere un calo del 50% nel 2000 con 11 positivi. Nel 2001 il numero di positivi è
pari a 29, con picco di positività nel 2002 e 2003 quando si registrano 63 e 62
positivi. Nel 2004 e 2005 i positivi si dimezzano rispetto ai due anni precedenti per raggiungere la punta di 73 positivi nel 2006.
Si registra quindi una prevalenza ad andamento sinusoidale con punte di minimo e di massimo ( 9% e 21%) tra il 1998 e il 2006, con un aumento negli
ultimi sei anni presi in esame dovuto, tuttavia, al maggior numero di test effettuati che sono passati da 108 nel 2000 a 460 nel 2006, con un aumento superiore al 400%.
Per Ehrlichia canis, Borrelia burgdorferi, Rickettsia rickettsii, Rickettsia conorii è stato effettuato il test del Chi Quadro considerando le positività nel periodo autunno-inverno e primavera-estate, dai risultati ottenuti è emerso che
non c’è stagionalità e che nell’arco dell’anno le malattie si presentano con frequenza simile.
Conclusioni. Trattandosi di uno studio retrospettivo ci sono dei limiti nell’uniformità dei dati.
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Ci sembra comunque utile avere registrato i dati raccolti, seppur relativi ad
una piccola realtà, poichè possono essere utili come temine di confronto per
analisi future.
Bibliografia
Acha P.N., Szyfres B., “Zoonoses et maladies transmissibles commune à l’homme et aux animaux. 2me edition”. O.I.E., 1989 Paris;
Baneth G., and Weigler B., “Retrospective case-control study of hepatozoonosis in dogs in Israel”. J. Vet. Int. Med., 1997, 11: 365-376;
Gavazza, A., Bizzeti, M., Papini, R., “Observation on dogs found naturally infected with Hepatozoon canis in Italy”. Revue de Medicine Veterinaire, 2003; 154 (8/9): 565-571;
Melgrati, E., Pilla, M., Casa, R., Borroni, G., Tiraboschi, M., Solari Basano, F.,Traldi, G., Genchi, C., 1997. “Sieroprevalenza di Rickettsia conorii in una popolazione canina residente
nel nord-ovest della provincia di Milano (ex U.S.S.L. 34) a seguito di alcuni casi autoctoni di infezione umana”.
Dott.ssa Stefania Pitarra, Via Taranto-Lecce 43, 74022 Fragagnano (TA), Italia
Cell. 320/4826878 - E-mail: ste_x@yahoo.it
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USEFULNESS OF A SEROLOGICAL SIMULTANEOUS
DIAGNOSIS OF FOUR CANINE VECTOR-BORNE
DISEASES IN DOGS
M. Renzi, DVM 1, L. Buratti, Biol 1, A. Santi, DVM 1, G. Galletti, Stat 1,
G. Paternoster, DVM 1, M. Tamba, DVM 1
1
Istituto Zooprofilattico Sperimentale della Lombardia e
dell’Emilia Romagna (IZSLER), Bologna, Italy
Purpose of the work. Lyme disease (LD), Canine Ehrlichiosis (CE), Rickettsiosis (Rickettsia conorii) (RC) and Leishmaniosis (CanL) are Canine Vector-Borne Diseases (CVBD) and zoonoses. These agents commonly produce
subclinical infection and the rate of seropositive animals is generally much
higher than the fraction developing clinical disease1.
CE-CanL co-infection was described in dogs2; co-infection with combinations
of Ehrlichia, Borrelia, Leishmania and Rickettsia species occurs in endemic
areas3, since the vectors’ activity (ticks and phlebotomine sand flies) and
transmission periods are similar. The role of ticks also for L. infantum transmission is reported4. Dogs with dual infections showed an increased presence
of clinical signs, suggesting a synergistic pathological effect between the etiological agents2. An early serological diagnosis in asymptomatic dogs is very
important to perform the proper therapy in infected dogs and/or to adopt preventive measures in exposed healthy dogs.
Aim of the work is to verify the usefulness of a serological simultaneous diagnosis of four CVBD (LD, CE, RC and CanL) in dogs, for early detection of
possible co-infections.
Materials and used methods. In 2012, 418 owned dogs from Emilia Romagna
region were sampled during the periodical clinical evaluation performed by a
private sector veterinarian. At the moment of sampling, a questionnaire was
filled in, to collect information regarding age, size, breed and possible clinical signs of CVBD. Sera were analyzed for CanL by IFAT5; cut-off was set at
1/40, IFAT titre of 1/40 or 1/80 were considered inconclusive, titres =1/160
were considered positive6. E, LD and RC were also diagnosed by IFAT using
kit Fuller® for E. canis (cut-off at 1:50), kit Fuller® for B burgdorferi (cut-off
at 1:80) and kit Biomerieux® for R. conorii (cut-off at 1:40). Exact Confidence
Intervals (CI) were calculated using binom.test function of R7.
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Outcomes. The examined population consisted in 140 hunting and 195 companion dogs; for the remaining 83 dogs this anamnestic information was unknown. Clinical signs were reported for 45 dogs only (10.8%).
57.4% of the tested dogs were positive for at least one test. Particularly, 200
dogs were positive for R. conorii (47.8%), 77 dogs for B. burgdorferi
(18.4%), 26 for L. infantum (6.2%) and finally 21 dogs were positive for E.
canis (5.0%).
The simultaneous use of four IFAT allowed detecting co-infections. On the
whole, 18.9% of the tested dogs showed more than 1 seropositivity. In detail,
we found 75 dogs (17.9%) with dual infection, 3 dogs (0.7%) with 3 simultaneous seropositivities and 1 dog positive to all 4 tests (0.2%). Considering the
attitudes of animals, 68.6% of hunting dogs were positive to at least 1 test as
compared to 46.7% of companion dogs.
29 of the 45 symptomatic dogs resulted seropositive for at least one infectious
agent (64.4%) and 14 dogs showed multiple seropositivities (31.1%). Among
the 373 asymptomatic dogs, 211 (56.6%) were seropositive for at least one
pathogen. The two more common co-infections were RC-LD, with a frequency of 8.6% and RC-CanL, 4.3%.
These results suggest a wide diffusion of CVBD.
Conclusions. As results of a heavy exposure to ticks, infection with multiple
pathogens can occur. The same tick species can be a vector for several
pathogens and infections can also be complicated by other arthropod-borne
diseases that share the tick biohabitat, such as CanL. Co-infections could partially explain variations in clinical presentation, pathogenicity and response
to therapy. Simultaneous testing of these four largely diffused CVBD helps
veterinarians in choosing the most proper therapeutic protocol and in adopting prophylactic measures to reduce the spread of zoonosis.
Bibliography
1. Dantas-Torres F, Felino de Brito EM, Brandao-Fihlo PS: Seroepidemiological survey on
canine Leishmaniasis among dogs from an urban area of Brazil. Vet. Parasitol. 2006, 140,
54-60.
2. Mekuzas Y, Gradoni L, Oliva G, Foglia Manzillo V, Baneth G: Ehrlichia canis and Leishmania infantum co-infection a 3–year longitudinal study in naturally exposed dogs.
Clin. Microbiol. Infect. 2009, 15-2, 30-31.
3. Shaw SE, Day MJ, Birtles RJ, Breitschwerdt EB: Tick-borne infectious diseases of dogs.
Trends in Parasitol. 2001, 17-2, 74-80.
4. Solano-Gallego L, Rossi L, Scroccaro AM, Montarsi F, Caldin M, Furlanello T, Trotta M:
Detection of Leishmania infantum DNA mainly in Rhipicephalus sanguineus male ticks
removed from dogs living in endemic areas of canine Leishmaniosis. Parasites and Vectors 2012, doi: 10.1186/1756-3305-5-98.
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5. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals ed. 2008 cap. 2.1.8.
6. Gradoni L, Gramiccia M, Khoury C, Maroli M: Linee guida per il controllo del serbatoio canino della leishmaniosi viscerale zoonotica in Italia. Rapporto ISTISAN 04/2012.
7. R Development Core Team: R: A language and environment for statistical computing.
2008, R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0.
Dott.ssa Maria Renzi - Istituto Zooprofilattico Sperimentale della Lombardia e
dell’Emilia Romagna (IZSLER)., Via Santo Stefano, 77, 40125 Bologna (BO), ITALY
Phone 051/226175 - Mobile 347/0458281 - E-mail: maria.renzi@izsler.it
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CANINE LEISHMANIASIS AND BIOLOGICAL NETWORKS:
A NEW APPROACH FOR AN OLD PROBLEM
D. Rispoli, DVM, DipSCPCA 1, G. Lubas, DVM, ECIM-CA, Full Professor
2
, N. Bernabò, DVM, PhD, Assistant Professor 3
1
Libero Professionista, Teramo, Italia
2
Dept. of Veterinary Sciences, University of Pisa, Via Livornese lato Monte,
San Piero a Grado, Pisa, Italia
3
Dept. of Comparative Biomedical Sciences, University of Teramo, Piazza
Aldo Moro 45, Teramo, Italia
Purpose of the work. Leishmaniasis is a widespread parasitic zoonoses1
which affect human and few other mammals including dogs and cats2,3.
Canids are the main reservoirs for the viscerotropic species in the Mediterranean, Asia, North Africa and South America1. Leishmaniasis represents indeed a serious problem for human health, with enormous social costs1,4,5. Despite the amazing efforts of scientific community, to date, really effective therapeutical resources are not still available. This could not be due to the scarcity of molecular data, but to the inability to manage them in explicative models. Thus, for the first time, we have adopted a very innovative biological networks-based approach to describe the relationship between the parasite and
the host. In particular, we described the recognition, binding and phagocytosis of Leishmania amastigote by monocyte as a network composed by nodes
(molecules involved) linked by edges (their interaction). Then, we carried out
a statistical analysis of network’s topology to obtain significative biological
inference.
Materials and used methods. We downloaded the data from KEGG Pathways
Menu (http://www.genome.jp/kegg-bin/show_pathway? org_name=cfa&
mapno=05140&mapscale=1.0&show_description=show), filtering the data
for Canis familiaris, pathway ID: cfa05140. The network (Leishmania Host
Interaction Network, LHIN) was created and analysed by Cytoscape 2.8.3
and Network Analyzer, and was classified on the basis of its topological parameters. Actually, it is possible to classify biological networks in different typologies according to these parameters. The most elementary characteristic
are the node degree, k, indicatig how many links the node has to other nodes,
and the node degree distribution, P(k), which represents the probability that
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a node has exactly k links. The network tendency to develop clusters of nodes
is expressed by the clustering coefficient CI = 2nI/k(k–1), where nI is the number of links connecting the kI neighbours of node I to each other. In random
networks, described by the Erdös–Rényi (ER)6 model, the node degrees follows a Poisson distribution and the clustering coefficient is independent by
nodes degree. Scale-free networks (Barabási–Albert, BA, model)7 are characterized by a power-law degree distribution of the number of links per node.
Thus, a relatively small number of nodes is highly connected (hubs) and most
of the nodes are scarcely linked. In addition, the clustering coefficient is independent by the number of links per node. In the hierarchical networks the
scale-free topology and the local clustering coexist.
Outcomes. We have found that LHIN is composed by 74 nodes and 85 edges.
The clustering coefficient was 0.018, the network diameter (i.e., the largest
distance between two nodes) was 8, the characteristic path length (i.e., the expected distance between two connected nodes) was 3.088, the averaged number of neighbours (i.e., the mean number of connections of each node) was
2.297. The most connected nodes were: nuclear factor kappa-light-chainenhancer of activated B cells (NFkB) extracellular-signal-regulated kinases
(ERK1/2) and protein kinase C (PKC), each 6 links; inducible nitric oxide
synthases (iNOS) gene and lipophosphoglycan (LPG), each 5 links. The LHIN
displayed two important characteristics:
- it is a random network, according to ER model;
- it is a directed network, having an input and an output terminal.
Conclusions. In our opinion, few conclusions can be drawn from these findings. The first could justify the failure of development of anti-leishmania
drugs. Indeed, random networks, differently from scale-free networks, are
highly resilient to attack (removal of most connected nodes)8, consequently, it
is very hard to destroy LHIN with a targeted drug therapy, as confirmed by a
computer simulation in which the removal of the most connected nodes did
not significatively affected the main topological indexes.
Secondly, since an attack strategy seems to be not effective, the only remaining way to destroy LHIN is to stop the flow of molecular event through the network. As strengthened by networks topology, LPG appears to be one of the
most connected nodes and, contemporaneously, it is the network input terminal. As a consequence, LPG seems to be a favourable target for drugs development against Leishmania. Interestingly LPG is described in literature as
the main responsible for the macrophages phagolysosome maturation inhibitions9,10,11 and in human Leishmania major LPG1- mutants show attenuated
virulence when compared with the wild type12,13.
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Bibliography
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Palatnik-de-Sousa CB et al. Parasit Vectors 2011 4: 197.
Quinnell RJ et al., Parasitology 2009 136:1915-1934.
Maia C et al., Trends Parasitol. 2011 27:341-3344.
Roberts M et al., J Glob Health 2011 1: 189–200.
Huda MM et al., BMC Public Health. 2012 12:1001.
Erdos P et al., Publ Math Debrecen 1959 6:290–297.
Barabási et al., Science 1999 286:509–512.
Guillaume et al., Proc. 8th Intl Conf Principles of Distributed Systems 2004 186-196.
Lodge R et al., Clin Immunol. 2005 114: 256-265.
Vinet AF et al., Microbiology, 2011, 157: 2619–2628.
Neda Moradin et al., Front in Cell and Infection Microb 2012 2:1-7.
Späth GF et al., PNAS 2000 97: 9258–9263.
Späth GF et al., PNAS 2003 100: 9536-9541.
Dott.ssa Daniela Rispoli, Via Balifieri 15, 64023 Mosciano Sant’angelo (TE), Italiana
Cell. 327-1837690 - E-mail: dane.rispoli@tiscali.it
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EVALUATION OF THE HUMORAL IMMUNE RESPONSE
AFTER THE FIRST ANNUAL CANILEISH®
BOOSTER VACCINATION
E. Sagols, DVM 1, F. Ferraz, DVM 2, E. Claret, PhD 1, D. McGahie, DVM 3
1
Bvt Groupe Virbac, La Seyne sur mer, France
2
Virbac Portugal Laboratorios LDA, Sintra, Portugal
3
Virbac SA, Carros, France
Purpose of the work. Canine Leishmaniosis caused by Leishmania infantum
is a severe zoonotic disease affecting dogs, highly endemic throughout the
Mediterranean basin. The treatment of this disease may be complex and often
non curative, so prevention is essential.
The recent availability of CaniLeish® (LiESP/QA-21), the first vaccine against L.
infantum for dogs in Europe, constitutes a new approach in leishmaniosis prevention. Using Excreted Secreted Proteins (ESP) of L. infantum and a specific adjuvant (QA-21), the vaccine stimulates the cell-mediated immune response of the dog against the parasite1,2. As part of this appropriate immune
response, there is also a temporary stimulation of antibodies (primarily IgG2)
against ESP and PSA (Parasite Surface Antigen)2 which may be detectable by
non-specific tests such as immunofluorescence antibody testing (IFAT). Previous investigations have assessed the consequences of a primary course of
the vaccine on serological tests: a transient increase of the IFAT titre was observed for up to four months3. These antibodies were not detected by the rapid immunochromatographic test Speed Leish K® 4 that uses a recombinant kinesin complex to detect anti-kinesin antibodies of L. infantum. Therefore, the
use of anti-kinesin antibodies allows discrimination between the antibodies
induced by vaccination after the primary course from those due to an infection3.
The aim of this study was to evaluate the serological immune response of the
dogs after the first annual CaniLeish® booster vaccination according to these
two methods.
Materials and used methods. 20 dogs having had a complete primary course
of vaccination with CaniLeish® one year before were included in the study.
The serological status of the dogs regarding leishmaniosis was evaluated just
before the CaniLeish® booster and 3 weeks to one month later with:
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- the rapid immunochromatographic test Speed Leish K®, to detect anti-kinesin antibodies against L. infantum.
- IFAT, with a positivity threshold titre at 1/100, to evaluate the total anti-L.
infantum antibody titre.
Vaccinations and tests were performed in accordance with the manufacturers’
instructions.
Outcomes. Before the CaniLeish® booster, all the dogs were negative with
Speed Leish K® and IFAT (one dog showed an IFAT titre at 1/50 considered
as non significant).
Concerning the results of the evaluation 3 weeks to one month after the booster, the distribution of the IFAT titres was as follows:
- 2 dogs remained negative,
- 3 dogs had an IFAT titre = 1/100,
- 2 dogs had an IFAT titre = 1/800.
All the dogs remained negative with Speed Leish K® after the booster.
Conclusions. Following the CaniLeish® booster, the IFAT titres varied from
negative to 1/800.
90% of the dogs had a titre less than or equal to 1/400, which corresponds to
4 times the positivity threshold of the IFAT used in this study, a level considered by the literature as representative of active leishmaniosis.
The IFAT titres obtained after the first annual booster vaccination with
CaniLeish® do not appear to be significantly different to those obtained after
the primary vaccination. It will be important to attempt to further follow these
dogs to see if these titres also drop to normal levels within 4 months as was
seen after the primary course. Speed Leish K® did not detect antibodies induced by the vaccination booster. The non-interference of these antibodies
with Speed Leish K® makes it tailored for use in any dogs presenting symptoms suggestive of leishmaniosis, whatever their vaccination status.
Bibliography
1. CaniLeish®: EPAR – Public assessment report – http://www.ema.europa.eu.
2. Moreno J, Vouldoukis I, Martin V, McGahie D, Cuisinier A-M, et al. - Use of a
LiESP/QA-21 Vaccine (CaniLeish®) Stimulates an Appropriate Th1-Dominated CellMediated Immune Response in Dogs. PLoS Negl Trop Dis, 2012 ; 6(6): e1683.
3. Sagols E, Martin V, Claret E, McGahie D, Cuisinier A-M and Gueguen S - Evaluation of
the humoral immune response after vaccination with LiESP/QA-21 (CaniLeish®): interest
of Leishmania specific anti-kinesin antibodies detection. WSAVA/FECAVA/BSAVA Congress. United Kingdom, Birmingham, 11th - 15th April 2012.
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4. Chêne J, Chabanne L, Morlet J and Bourdoiseau G - Comparison of a rapid immunochromatographic test (Speed Leish KTM, BVT) with immunofluorescence assay for the
detection of anti-Leishmania infantum antibodies in dogs. Proceedings of the 2nd International Congress on Canine Leishmaniasis. Pisa, Italy, 17 - 18 April 2010.
Dott.ssa Emmanuelle Sagols - Bvt Groupe Virbac, 285 Avenue De Rome,
83500 La Seyne Sur Mer, France - Phone +33494105894 - E-mail: esagols@bvt.fr
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AUTOCHTHONOUS CASES OF DIROFILARIA
REPENS IN GERMANY, AN EXAMPLE
FOR THE SPREADING OF A VECTOR BORNE DISEASE
AS A RESULT OF CLIMATE CHANGE
R. Sassnau, Dr. med. vet. 1, C. Genchi, Prof. 2
1
Small animal practice, Berlin, Germany
2
Dipartimento di Scienza Veterinarie e Sanità Pubblica,
Università degli Studi di Milano, Milan, Italy
Purpose of the work. Case reports of cutaneous dirofilariosis in dogs as well
as in humans living in North Europe have usually been limited to imported
“travel disease” (Keller et al., 2007). In the past decade a lot of autochthonous cases of cutaneous dirofilariosis were reported out of locations towards
previously non-endemic areas (Cieleka et al., 2012; Pantchev et al., 2009).
In dogs, D. repens is regarded as less pathogenic but nevertheless an important agent of disease in humans.
Apart from subconjunctival localizations where the parasite can be easily observed, other diagnostic tools are not fully reliable and serology is not specific. As a consequence, cases of misdiagnosis and incorrect treatment are not
infrequent in men.
The aim of this study was to examine whether climate change could be responsible for local changes making possible the extrinsic incubation of the
parasite with the risk of a possible endemisation in previously infection-free
regions like the state of Brandenburg in comparison to the endemic area
around Karlsruhe (Baden-Wuertemberg) [2012; Pantchev et al., 2009].
Materials and used methods. The records of the average daily temperatures
from the German Weather Service (DWD, 2012) were obtained for the stations of Neuruppin, Potsdam, Angermünde and Berge in the state of Brandenburg (North-East Germany) and the station of Karlsruhe (South-West
Germany).
Methods used. Daily mean temperatures throughout May 1st and October
15th assumed as the potential local mosquito activity season were used and
the larval development unit (DU) for D. repens was assessed as follows: DU
is defined as the difference between the average daily temperature, providing
=15 °C, and the limit of 14 °C. At least 130 DU are necessary for one extrin-
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sic development of microfilariae to infective larvae. The 130 DU have to be
achieved within 30 days (Slocombe et al, 1989) [hereafter referred as 130
LDU/30]. The linear regression of the sum of days for each year with >130
LDU/30 has been calculated. Data was generated with MS Excel.
Outcomes. The analysis of the mean daily temperatures showed a rising trend
during the years 1960-2012 for all the Brandenburg stations as well as in
Karlsruhe where, however, the mean temperatures were constantly higher by
about one °C.
The number of days with >130 LDU/30 showed an increasing trend. For
Brandenburg stations, the graphs of the 130 LDU/30 were close together.
The requirement of >130 LDU/30 was not fulfilled in 12 of 52 observed
years in Neuruppin as a representative for the state of Brandenburg. In
contrast to Brandenburg, no year with less than 130 LDU/30 was found in
Karlsruhe.
Temperature is a factor that dictates the mosquito vector ability to complete
the extrinsic development of D. repens, and this was possible in the past 12
years in the state of Brandenburg as a result of climate change.
In 2007 the first evidence of autochthonous cutaneous dirofilariasis in 5 dogs
from a sled dog kennel in the Havelland (district in the state of Brandenburg)
was found. 11 new autochthonous cases were diagnosed in the same kennel in
2012. The finding of these 16 autochthonous cases associated with sufficient
temperature for larval extrinsic development and an increasing infection
pressure due to travelling pets might made the endemisation of D. repens possible in the state of Brandenburg.
Conclusions. Climate change with an increase in average temperature may
be responsible for propagation of temperature dependent vectors and/or vector-associated pathogens in regions that were previously not endemic. The
analysis of climate data of Brandenburg state had showed that the extrinsic
development of D. repens, the causative agent of subcutaneous dirofilariosis,
was possible from the years 2001 to 2012. This finding, associated to the
movement of infected dogs and their relocation from endemic European areas
to Germany makes possible a rapid endemisation of the infection and highlight the risk for human health, being D. repens a frequent cause of zoonotic
infections.
Bibliography
Cieleka D, Žarnowska-Prymek H, Masny A, Wesolowska M, Golab E (2012) Human dirofilariosis in Poland: the first autochthonous infections with D. repens. Ann Agric Environ
Med 19(3): 445-50
DWD. Deutscher Wetterdienst, Offenbach/Main www.dwd.de
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Keller L, Hartmann K, Wess G (2007) Fallbericht und Literaturübersicht kutaner Dirofilariose.
Tierärztl Prax 35(K):31-4.
Pantchev N, Norden N, Lorentzen L, Rossi M, Rossi U, Brand B, Dyachenko V (2009) Current surveys on the prevalence of Dirofilaria spp. in dogs in Germany. Parasitol Res 105
Suppl 1: 63-74.
Slocombe JOD, Surgeoner GA, Srivastava B (1989) Determination of heartworm transmission
period and its use in diagnosis and control. Proc Heartworm Symp ’89. America Heartworm Society, Washington, DC: 19-26.
Dr. Reinhold Sassnau, Hasenheide 65, D 10967 Berlin, Germany
E-mail: sassnau@web.de
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ESTRATTI
RELAZIONI AZIENDALI
COMPANY RESEARCH ABSTRACTS
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Giada Bagnagatti De Giorgi
Med Vet, Milano
Coinvolgimento renale in corso
di Leishmaniosi canina
Venerdì, 8 Marzo 2013, ore 12.35
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La Leishmaniosi canina (Lcan) è una malattia infettiva sistemica a carattere
zoonosico che colpisce diversi mammiferi, tra cui il cane, causata da protozoi flagellati del genere Leishmania e che si trasmette prevalentemente durante il pasto di
sangue di un insetto vettore appartenente al genere Phlebotomus (Vecchio Mondo)
o Lutzomyia (Nuovo Mondo). Il cane è un ospite recettivo ed è considerato il principale reservoir d’infezione (Solano-Gallego et al, 2009). Le alterazioni cliniche e
clinico patologiche che si riscontrano in soggetti affetti da Lcan sono estremamente varie come conseguenza dei numerosi meccanismi patogenetici, dei differenti
organi colpiti e dalla differente risposta immunitaria messa in atto dall’ospite (Solano-Gallego et al, 2009). In particolare è la risposta immunitaria umorale che, oltre ad avere scarso potere protettivo, porta alla produzione di immunocomplessi
circolanti responsabili di alterazioni patologiche d’organo come vasculite, uveite,
poliartrite e glomerulonefrite (Solano-Gallego et al, 2009).
Il rene è uno degli organi maggiormente interessati, talvolta anche l’unico
organo coinvolto in soggetti malati (Ciaramella et al, 1997). Le lesioni renali
primarie sono rappresentate da glomerulopatie proteinuriche che, nel tempo favoriscono la comparsa di un danno tubulo interstiziale con finale evoluzione
della nefropatia verso l’insufficienza renale. Istologicamente tali lesioni si possono classificare in glomerulonefriti mesangiali, membranose, membranoproliferative e focali segmentali (Zatelli et al, 2003). Risulta dunque di fondamentale importanza valutare costantemente la funzionalità renale dei cani leishmaniotici durante e dopo la terapia. La valutazione della funzionalità renale
e la stadiazione mediante l’applicazione delle raccomandazioni dell’International Renal Interest Society (IRIS) basate principalmente su valutazione di
creatinina sierica, proteinuria e ipertensione sono di fondamentale importanza
per controllare l’evoluzione del danno glomerulare e per associare alle terapie
mirate al trattamento della Lcan (Oliva et al, 2010) terapie per il controllo dell’evoluzione del danno renale al fine di poter migliorare la qualità e le prospettive di vita del soggetto leishmaniotico (Solano-Gallego et al, 2009).
BIBLIOGRAFIA
Ciaramella P, Oliva G, Luna RD, Gradoni L, Ambrosio R, Cortese L, Scalone A, Persechino A.
A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by
Leishmania infantum. Vet Rec. 1997 Nov 22;141(21):539-43.
Oliva G, Roura X, Crotti A, Maroli M et al. Guidelines for treatment of leishmaniasis in dogs.
J Am Vet Med Assoc. 2010 Jun 1;236(11):1192-8.
Solano-Gallego L, Koutinas A, Miró G, Cardoso L et al. Directions for the diagnosis, clinical
staging, treatment and prevention of canine leishmaniosis. Vet Parasitol. 2009 Oct 28;165
(1-2):1-18.
Zatelli A, Borgarelli M, Santilli R, Bonfanti U, Nigrisoli E, Zanatta R, Tarducci A, Guarraci A.
Glomerular lesions in dogs infected with Leishmania organisms. Am J Vet Res. 2003 May;
64(5):558-61.
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Gaetano Oliva
Med Vet, Dr Ric, Napoli
CVBD Clinical Center: un supporto
al clinico per la gestione delle
malattie trasmesse da vettore
Venerdì, 8 Marzo 2013, ore 17.20
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SCOPO DEL LAVORO
Le malattie trasmesse da artropodi vettori nel cane (Canine Vector Borne
Diseases - CVBD) costituiscono uno dei capitoli più difficili ed affascinanti
della medicina interna, in virtù della complessità diagnostica che spesso tali
malattie richiedono. Lo scopo del presente lavoro è la descrizione di uno strumento telematico interattivo, il CVBD Clinical Center.
MATERIALI E METODI
Il CVBD Clinical Center è un supporto telematico interattivo, messo a disposizione dei Medici Veterinari italiani, per offrire loro uno strumento di facile consultazione sia per la possibile risoluzione di casi clinici di non semplice gestione, ma anche per avere risposte immediate a dubbi riguardanti la
diagnosi, la gestione clinica, la terapia, il monitoraggio e la prevenzione delle CVBD. Il Centro è costituito da una pagina web (www.advantix.it) alla
quale i Medici Veterinari dopo una rapida e facile registrazione possono accedere per formulare i propri quesiti ad alcuni esperti del settore. Il sistema,
oltre alla formulazione di domande, consente il caricamento di cartelle cliniche, esami diagnostici ed immagini, utili all’interazione e allo scambio di informazioni tra Colleghi.
RISULTATI
Il CVBD Clinical Center, inaugurato nel mese di settembre 2012, ha già
ricevuto numeroso contatti e richieste da parte di Medici Veterinari di diverse
regioni, che ha comportato non solo al chiarimento di alcuni dubbi clinicodiagnostici, ma anche e soprattutto alla crescita professionale sia dei richiedenti che degli esperti chiamati in causa.
CONCLUSIONI
Nella presente relazione, l’Autore oltre a dimostrare ai Colleghi presenti
la funzionalità pratica e la semplicità dell’uso di tale strumento, discuterà su
un caso clinico di Leishmaniosi canina caratterizzato da episodi sincopali, la
cui risoluzione diagnostica ha visto una proficua collaborazione tra il Medico veterinario chiamato a gestire il caso e l’esperto di turno chiamato a collaborare.
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BIBLIOGRAFIA
1. Incidence and time course of Leishmania infantum infections esamine by parasitological,
serologic, and nested-PCR techniques in a cohort of naïve dogs exposed to three consecutive transmission seasons. Oliva G, Scalone A, Foglia Manzillo V, Gramiccia M, Pagano A, Di Muccio T, Gradoni L. J Clin Microbiol. 2006 Apr;44(4):1318-22.
2. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L,
Bourdeau P, Oliva G, Baneth G. Vet Parasitol. 2009 Oct 28;165(1-2):1-18. Epub 2009
Jun, 6.
3. Ehrlichia canis and Leishmania infantum co-infection: a 3-year longitudinal study in naturally exposed dogs. Mekuzas Y, Gradoni L, Oliva G, Foglia Manzillo V, Baneth G. Clin
Microbiol Infect. 2009 Mar 26.
Dott. Gaetano Oliva - Dipartimento di Medicina Veterinaria e Produzioni Animali,
Via F. Delpino, 1, 80137 Napoli (NA), Italia - Tel. 0812536012 - Cell. 347 3096203
E-mail: gaeoliva@unina.it
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Luigi Gradoni
Marco Melosi
Scien Biol, Istituto
Superiore di Sanità,
Roma
Med Vet, Cecina (LI)
SCALIBOR Map Update:
Mappatura Nazionale zone a
Rischio Leishmaniosi 2013
Sabato, 9 Marzo 2013, ore 12.50
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In Italia dove 1 famiglia su 4 possiede un cane, i focolai in aree tradizionalmente non endemiche continuano ad aumentare.
Anno dopo anno, la Leishmaniosi si sta espandendo dai territori tradizionalmente endemici all’Italia centrale, verso le aree collinari e pedemontane
interne di Toscana, Umbria e Marche, ma anche verso il Nord Italia, che prima non era considerato endemico per questa malattia. Secondo l’analisi degli
accertamenti condotti negli ultimi 6 anni dagli Istituti Zooprofilattici, dalle
Università e dall’Istituto Superiore di Sanità (ISS) su oltre 500.000 campioni
sierologici, sono oltre 150 i comuni riscontrati positivi in Piemonte e Valle
d’Aosta, una trentina in Lombardia, e 80 tra Veneto, Trentino e Friuli-Venezia Giulia. La presenza di Leishmaniosi canina endemica è stata accertata in
oltre 2.700 Comuni Italiani, pari a circa il 34% del totale.
Proprio alla luce di questa forte espansione delle zone endemiche, l’Organizzazione Mondiale della Sanità ha rivisto le Linee Guida, adeguandole all’aumentata prevalenza della malattia, e anche l’Europa è corsa ai ripari lanciando, attraverso l’European Centre for Disease Prevention and Control, un
programma dedicato alle malattie trasmissibili da vettori, identificando nella
leishmaniosi una patologia prioritaria. L’ISS è stato incaricato dalle Autorità
europee affinchè coordinasse a livello nazionale un progetto di mappatura sulle zone a rischio di leishmaniosi, tenendo conto che le infezioni del cane rappresentano la più efficace sentinella epidemiologica di trasmissione endemica in un territorio.
Nel 2010 i dati, frutto di questa prima indagine epidemiologica, sono stati valorizzati per la prima volta, con il contributo di MSD Animal Health, in
una mappa cartografica (SCALIBOR® Map) che evidenzia tramite un codice
colore (rosso) le aree in cui è stato possibile accertare la presenza autoctona
di leishmaniosi.
Il progetto SCALIBOR® Map Update, partito il 16 gennaio e “live” fino al
31 maggio 2013, si pone come obiettivo quello di verificare la situazione epidemiologica nei comuni che allo stato attuale sono indicati sulla SCALIBOR®
Map come “bianchi”, ovvero “esenti da leishmaniosi”. Questi comuni comprendono territori nei quali la malattia può ragionevolmente essere considerata esente per caratteristiche geo-climatiche sfavorevoli. Tuttavia esistono anche numerosissimi territori individuati dall’ISS mediante incrocio con i dati
del “Geographic Information System” (1.116 Comuni Italiani) che sono a “rischio epidemiologico”, ovvero:
“Territori in cui non essendo stato possibile fino ad oggi documentare la
presenza di soggetti autoctoni infetti, presentano tuttavia condizioni geo-climatiche compatibili con la presenza del vettore”.
In considerazione della valenza sanitaria e dell’evidente interesse epidemiologico del progetto SCALIBOR Map Update, ANMVI ha deciso di farsi
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promotrice del progetto presso i Medici Veterinari, promuovendo insieme a
MSD Animal Health anche una campagna di sensibilizzazione a livello di
proprietari di cani, sul rischio della diffusione della leishmaniosi a livello nazionale. Le modalità di adesione dei Medici Veterinari al progetto SCALIBOR Map Update sono libere, gratuite, volontarie e aperte a tutti i colleghi
che esercitano la loro professione nei territori comunali valutati a “rischio epidemiologico” o considerati “esenti”.
I Medici Veterinari che decideranno di aderire al progetto dovranno inviare all’ISS un campione di siero prelevato da un cane sospetto infetto o ammalato per il quale viene garantita l’autoctonia del caso all’interno del comune considerato. In caso di accertamento positivo, e successivamente ad ulteriori indagini, il comune in questione potrà essere inserito a pieno titolo nella
lista dei territori “infetti”.
MSD Animal Health, con la collaborazione e il supporto di ANMVI ha
pertanto istituito un sito di servizio per tutti i veterinari (http://vet.scalibormap.it) i quali potranno navigare per conoscere se la Regione, la Provincia e
il Comune di loro interesse risulta “esente”, “infetto” o “sospetto/da indagare” per l’accertamento della leishmaniosi del cane.
La raccolta dei nuovi dati e la loro valorizzazione avverranno tramite supporti informatizzati di dialogo e interscambio tra Medici Veterinari aderenti e
gli attori del progetto (ISS, ANVVI e MSD Animal Health).
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